Labelling compounds and their use in assays

ABSTRACT

The invention provides monoferrocenyl compounds of general formula I. The invention also provides substrates labelled with the compounds, functionalised derivatives of the compounds and methods of using the compounds, functionalised derivatives and labelled substrates in electrochemical assays.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/026,454 entitled “Labelling Compounds and Their Use in Assays,” filedon Mar. 31, 2016, which is a national stage application, filed under 35U.S.C. § 371, of International Application No. PCT/GB2014/053031, filedon Oct. 8, 2014, and claims the benefit of, and priority to, GB PatentApplication No. 1413931.5 filed on Aug. 6, 2014, and GB PatentApplication No. 1317787.8 filed on Oct. 8, 2013, the complete contentsof which are hereby incorporated herein by reference in their entiretyfor all purposes.

FIELD OF THE INVENTION

The invention relates to ferrocenyl labelling compounds and the use ofsuch compounds in electrochemical assays and electrochemical detectionmethods.

BACKGROUND OF THE INVENTION

The detection of certain biological molecules plays an important part inmany aspects of life. For example, in the medical field, there is anever-present need to detect bacterial or viral pathogens, or biologicalmolecules. Other fields in which sensitive assays are essential includethe food and beverage industries. One method of detection involves theuse of electrochemically active compounds. The application ofelectrochemical detection has a number of advantages over other methods,such as fluorescent detection. Electrochemical detection has thepotential for very high levels of sensitivity and exhibits a widerlinear dynamic range than fluorescence. Furthermore, there is norequirement for samples to be optically clear. There is also lessinterference from background contaminants (many biological samplesauto-fluoresce).

WO03/074731 discloses electrochemically active markers and methods ofprobing for a nucleic acid. The methods involve contacting a nucleicacid solution with an oligonucleotide probe attached to anelectrochemically active marker. The probe is caused to at leastpartially hybridise with any complementary target sequence which may bepresent in the nucleic acid solution. Following enzymatic degradation ofthe nucleic acid probe, information is electrochemically determinedrelating to the marker.

Hillier et al (Bioelectrochemistry 63 (2004) 307-310) describes the useof ferrocene urea compounds as labels in pulse electrochemical methodsfor the electrochemical discrimination between a labelledoligonucleotide and an enzyme digested labelled oligonucleotide.

WO2005/005657 discloses further electrochemically active markers andmethods of detecting protease activity. The methods involve contacting asample solution with a protease substrate attached to anelectrochemically active marker, providing conditions under which anyprotease present in the sample can degrade the protease substrate.Following degradation, information is electrochemically determinedrelating to the marker.

WO2012/085591 and WO2013/190328 describe certain diferrocenyl compoundsfor use as electrochemical labels.

There is a continuing need to develop labels that enable detection ofthe presence of biological substrates or indicators, for example,nucleic acids or amino acids, in low concentrations. In particular,there is a continuing need for new labels with different oxidationpotentials and/or with different chemical or physical properties therebywidening the range of possible assays available and increasing the scopefor the development of multiplex reactions. Furthermore there is a needfor electrochemically active compounds which can be used as internalcontrols in assays. Such compounds need to give robust, consistentelectrochemical responses.

SUMMARY OF THE INVENTION

The present invention provides new ferrocenyl labelling compounds,functionalised derivatives of the compounds and substrates labelled withthe compounds. The compounds of this invention have been found to beeffective labels for use in electrochemical assays. The compounds of theinvention have also been found to give robust, consistentelectrochemical responses with oxidation potentials between −150 mV and584 mV, so they may be useful as internal controls in assays. When usedas probes in assays, the compounds of the invention give consistent andreproducible peak heights.

Furthermore, the compounds of the invention exhibit a largeelectrochemical range, allowing excellent tuning for an internal controllabel to be in a “clean” area of the voltammogram i.e. in an arearemoved from other peaks. Thus, the compounds of the invention are veryuseful in multiplex assays.

The compounds and labelled substrates of the invention may be used inany other electrochemical technique in which their electrochemicalcharacteristics can be utilised to derive information about the labelsor their environment.

An embodiment of the invention provides a compound having generalformula I

-   -   wherein:        -   each X substituent is independently selected from halo,            vinyl, alkyl, cycloalkyl, SiR₃, SnR₃, PR₂, P(O)R₂, SR,            S(O)R, SO₂R, aryl, heteroaryl, CHO, CO₂R, CN and CF₃;        -   each R is independently selected from alkyl, cycloalkyl,            aryl and heteroaryl;        -   Y is a spacer;        -   Z is a spacer;        -   L is a linker group;        -   a is 0, 1, 2, 3 or 4;        -   b is 0, 1, 2, 3, 4 or 5; and    -   vinyl, alkyl, cycloalkyl, alkylene, aryl and heteroaryl may        optionally be substituted with 1, 2 or 3 substituents        independently selected from unsubstituted alkyl, OH, CN,        fluorine, chlorine, bromine and iodine.

The labelling compounds of the invention and the labelled substratesderived therefrom offer characteristics which make them usefulcomplements to previously known labelling compounds, permitting a widerspectrum of applications. For example, the compounds and labelledsubstrates of the invention may offer additional opportunities foravoidance of conditions under which measurement potential may becompromised by interference with impurities that may be present. Thecompounds and labelled substrates of the invention also offer differingelectrochemical potential values, potentially allowing greaterflexibility in multiplex assays.

Electrochemical activity of a marker is primarily modulated by thesubstituents on the ferrocenyl group. Therefore choice of X, a and b canallow the electrochemical potential of the compound to be selectedappropriately. Further fine tuning can be achieved by the choice of Yand Z.

In an embodiment, the invention relates to a compound of general formulaIA

-   -   wherein:        -   each X substituent is independently selected from halo,            vinyl, alkyl, cycloalkyl, SiR₃, SnR₃, PR₂, P(O)R₂, SR,            S(O)R, SO₂R, aryl, heteroaryl, CHO, CO₂R, CN and CF₃;        -   each R is independently selected from alkyl, cycloalkyl,            aryl and heteroaryl;        -   A is O, B is CH₂ and c is 1, or        -   A is CH₂, B is O and c is 2;        -   a is 0, 1, 2, 3 or 4;        -   b is 0, 1, 2, 3, 4 or 5; and    -   vinyl, alkyl, cycloalkyl, alkylene, aryl and heteroaryl may        optionally be substituted with 1, 2 or 3 substituents        independently selected from unsubstituted alkyl, OH, CN,        fluorine, chlorine, bromine and iodine.

In another embodiment, the invention provides a compound of generalformula IB

-   -   wherein:        -   each X substituent is independently selected from halo,            vinyl, alkyl, cycloalkyl, SiR₃, SnR₃, PR₂, P(O)R₂, SR,            S(O)R, SO₂R, aryl, heteroaryl, CHO, CO₂R, CN and CF₃;        -   each R is independently selected from alkyl, cycloalkyl,            aryl and heteroaryl;        -   A is O, B is CH₂ and c is 1, or        -   A is CH₂, B is O and c is 2; and    -   vinyl, alkyl, cycloalkyl, alkylene, aryl and heteroaryl may        optionally be substituted with 1, 2 or 3 substituents        independently selected from unsubstituted alkyl, OH, CN,        fluorine, chlorine, bromine and iodine.

Preferably in the compounds of formula I, Y and Z are both alkylene,either or both of which is/are optionally be substituted with 1, 2 or 3substituents independently selected from unsubstituted alkyl, OH, CN,fluorine, chlorine, bromine and iodine. More preferably Y isstraight-chained alkylene which may be substituted with 1, 2 or 3substituents independently selected from OH, CN, fluorine, chlorine,bromine and iodine and Z is Cl or C3-C8 alkylene which may besubstituted with 1, 2 or 3 substituents independently selected fromunsubstituted alkyl, OH, CN, fluorine, chlorine, bromine and iodine.

In the compounds of the invention, L is any linker group suitable foreffecting linkage to the substrate either directly or via afunctionalising group as described herein. L is advantageously a linkergroup comprising an oxygen atom. L is preferably a hydroxy group or aprotected hydroxy group. Most preferably L is a hydroxy group.

In the compounds of the invention, ferrocenyl may have only one Xsubstituent, such that a+b=1. Ferrocenyl may have no substituent on thedistal cyclopentadienyl ring, such that b is 0. Ferrocenyl may have onlyone substituent on the proximal cyclopentadienyl ring, such that a is 1.

In an embodiment of the compounds of the invention, each X may beindependently selected from halo, vinyl, alkyl, cycloalkyl, SiR₃, SnR₃,P(O)R₂, SR, S(O)R, SO₂R, aryl, heteroaryl, CHO, CO₂R, CN and CF₃. Inanother embodiment of the compounds of the invention, each X may beindependently selected from halo, vinyl, SR, S(O)R, alkyl, P(O)R₂,S(O)₂R, SiR₃. In a particular embodiment, each X is independentlyselected from SR, S(O)R and S(O)₂R. In another embodiment, a is 4, b is5 and each X is methyl.

The compounds of the invention are labelling compounds suitable to formlabelled substrates. Attachment of the compounds to a substrate may bedirect (e.g. via L) or via a functionalising group, preferably via aphosphoramidite group. Thus, in an embodiment, the invention providescompounds which are functionalised derivatives of the compounds of theinvention. Preferably, the functionalised derivatives comprise afunctionalising moiety selected from succinimidyl ester groups,phosphoramidite groups, maleimide groups, biotin and azide groups. In aparticular embodiment, the functionalising moiety is a phosphoramiditegroup.

In another embodiment the invention provides substrates labelled with acompound of the invention. Substrates that may be labelled includenucleic acids, amino acids, polypeptides, carbohydrates and derivativesor synthetic analogues of any of those molecules. Other substrates thatmight be labelled include latex/paramagnetic particles.

In a preferred embodiment, the substrate is a nucleic acid. Preferablythe nucleic acid has a sequence which is complementary to a sequence ina microorganism selected from the group consisting of Chlamydiatrachomatis, Trichomonas vaginalis, Neisseria gonorrhoeae, Mycoplasmagenitalium and methicillin resistant Staphylococcus aureus. In anembodiment the substrate is not adenosine.

In another preferred embodiment, the substrate is an amino acid,polypeptide or carbohydrate; or a nucleic acid comprising at least 2nucleotides.

An assay kit for determining the presence of an assay target, whereinthe assay kit comprises a labelled substrate of the invention, is alsoprovided.

Another embodiment provides the use of a compound of any of theembodiments of the invention as a label in an electrochemical assay. Ina particular embodiment, the assay is for detecting an electrochemicallylabelled substrate. More particularly, the assay is for determining theamount of an electrochemically labelled substrate. For example, thecompounds of the invention may find use in a method as described inWO03/074731 or in a method as described in WO2005/005657.

Another embodiment provides a method for the manufacture of afunctionalised derivative of a compound of formula I, comprisingreacting a compound of formula I with a functionalising compound. In aparticular embodiment the functionalising compound comprises aphosphoramidite group.

Also provided is a method for the manufacture of a labelled substratecomprising reacting a compound of any of the embodiments of theinvention with a substrate to obtain a labelled substrate.

Another embodiment provides a method of detecting a nucleic acid in asample comprising contacting a nucleic acid with a complementary nucleicacid probe under conditions to allow hybridization between the probe andthe nucleic acid, wherein the probe is labelled with a compound of anythe embodiments of the invention. The method can include the furtherstep of measuring the electrochemical activity of the compound labellingthe probe. Optionally the method comprises the step of selectivelydegrading the either hybridised or unhybridised probe, prior to themeasuring step. Selective degradation of a hybridised probe may beeffected by a double strand specific exonuclease enzyme. Theelectrochemical activity of the compound of the invention may bedependent either quantitatively or qualitatively on the extent ofdegradation of the probe. Optionally the nucleic acid is amplified (forexample by PCR or another nucleic acid amplification technique) prior tocontacting it with the probe.

Another embodiment provides a method of detecting a substrate labelledwith a compound of any of the embodiments of the invention, comprisingthe step of measuring the electrochemical activity of the compound. Inan embodiment, there is used an assay device comprising at least twolabels, each label comprising a compound or labelled substrate accordingto the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows voltammograms obtained from the multiplex PCR assaydescribed in example 22 below.

FIG. 2 shows a graph cataloguing the use of3-(nonamethylferrocenylmethoxy)propan-1-ol (example compound 2) as aprobe in a series of detection assays, as described in example 23.

DETAILED DESCRIPTION

The term “alkyl” refers to straight-chain alkyl groups having from 1 to8 carbon atoms, preferably from 1 to 6 carbon atoms, and more preferablyfrom 1 to 4 carbon atoms and branched chain alkyl groups having from 3to 8 carbon atoms, preferably from 3 to 6 carbon atoms. Illustrativealkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl andt-butyl.

The term “cycloalkyl” refers to saturated or partially saturatedcarbocylic ring having from 3 to 8 ring members, preferably from 3 to 6ring members. One or more ring members may be selected from heteroatomssuch as oxygen, sulphur and nitrogen. Illustrative cycloalkyl groupsinclude cyclohexyl, cyclopentyl, piperidinyl and morpholinyl.

The term “alkylene” refers to a bivalent straight-chained alkyl radicalhaving from 1 to 8 carbon atoms, preferably from 1 to 6 carbon atoms,more preferably having 1 to 4 carbon atoms or a bivalent branched-chainalkyl radical having 2 to 6 carbon atoms, preferably 3 to 4 carbonatoms.

The term “alkenyl” refers to straight- or branched-chain alkenyl groupshaving from 2 to 6 carbon atoms, more preferably from 2 to 4 carbonatoms. Illustrative alkenyl groups include ethenyl, propenyl andbutenyl.

The term “aryl” refers to an unsaturated, aromatic monocyclic orbicyclic ring having from 5 to 10 carbon members. Illustrative arylgroups include phenyl and napthalenyl.

The term “heteroaryl” refers to an aromatic monocyclic or bicyclicaromatic ring system having 5 to 10 ring members and which containscarbon atoms and from 1 to 4 heteroatoms independently selected fromheteroatoms such as oxygen, sulphur and nitrogen. Illustrativeheteroaryl groups include furanyl, imidazolyl and thiazolyl.

“Halo” or “halogen” refers to fluorine, chlorine, bromine and iodine.

The term “proximal cyclopentadienyl ring” refers to the cyclopentadienylring to which the spacer group Y is attached. The term “distalcyclopentadienyl ring” refers to the cyclopentadienyl ring to which thespacer group Y is not attached.

With reference to substituents, the term “independently” refers to thesituation where when more than one substituent is possible, thesubstituents may the same or different from each other.

Except where the contrary is apparent from the context, references tothe term “substrate” are to be understood to include both naturallyoccurring substrates and synthetic substrates. References tocarbohydrates, nucleic acids, amino acids and polypeptides, are to beunderstood as referring to naturally occurring or syntheticcarbohydrates, nucleic acids, amino acids and polypeptides.

The term “polypeptide” refers to any chain of amino acids linked bypeptide bonds comprising two or more amino acid residues, such as adipeptide or a complex protein.

The term “nucleic acid” refers to a molecule comprising one or morenucleic acid residues and includes nucleotides, nucleosides,oligonucleotides and polynucleotides, and includes DNA and RNA. Thenucleic acid may comprise 1 to 50 nucleotides, more preferably from 2 to40 nucleotides especially from 15 to 35 nucleotides, with from 18 to 30nucleotides being especially preferred. For some applications, shorterlength substrates may be useful, for example nucleic acid with from 1 to14 nucleotides, more preferably from 2 to 10 nucleotides. Nucleotidesmay be selected from adenosine, thymidine, guanosine, cytidine oruridine nucleotides. When the nucleic acid is attached to a labelprovided herein, it is preferably attached through a group attached tothe ribose or deoxyribose group of a nucleotide, for example in the 2′,3′ or 5′ position, such as through an oxygen or nitrogen atom. Mostpreferably, the nucleic acid is attached at the 3′ or 5′ position of anucleotide, for example at the 5′ position. The sequence of the nucleicacid portion of the substrate is preferably such that the substrate isable to hybridise with a complementary target sequence and thus be usedas a probe in a molecular biological technique, for example, one of thenucleic acid detection techniques disclosed herein.

The term “carbohydrate” refers to a molecule comprising one or moresaccharide residue and includes monosaccharides, oligosaccharides, andpolysaccharides.

Substrates can be single nucleotides and single amino acids. In the caseof an assay relying upon cleavage of a substrate, for example by anenzyme, a single amino acid or nucleotide may be regarded as a substratebecause, although it lacks an internal bond capable of being cleaved byan enzyme, such a bond may be formed through the attachment of a marker.In an embodiment the substrate is not adenosine.

Where derivatives of naturally occurring substrates are referred toherein, those derivatives may be naturally occurring derivatives orsynthetic derivatives of the substrate.

References to the term “hybridise” in the context of nucleic acids willbe understood to mean specific binding of a first nucleic acid to asecond nucleic acid of complementary sequence. It will also beunderstood that in order for hybridisation to occur the complementarityof nucleic acid sequences is not required to be total. Hybridisationincludes complementary binding that includes base mis-match to theextent that such mis-match shall not materially reduce the efficiency ofthe methods described.

A compound of the invention as described above may be attached directlyto a substrate, or after functionalisation of the compound and/or orafter derivatisation of the substrate.

A functionalised derivative according to the invention may be a compoundaccording to formula II:

-   -   wherein:        -   each X substituent is independently selected from halo,            vinyl, alkyl, cycloalkyl, SiR₃, SnR₃, PR₂, P(O)R₂, SR,            S(O)R, SO₂R, aryl, heteroaryl, CHO, CO₂R, CN and CF₃;        -   each R is independently selected from alkyl, cycloalkyl,            aryl and heteroaryl;        -   Y is a spacer;        -   Z is a spacer;        -   F is a functionalising group;        -   a is 0, 1, 2, 3 or 4;        -   b is 0, 1, 2, 3, 4 or 5; and    -   vinyl, alkyl, cycloalkyl, alkylene, aryl and heteroaryl may        optionally be substituted with 1, 2 or 3 substituents        independently selected from unsubstituted alkyl, OH, CN,        fluorine, chlorine, bromine and iodine.

F may be derived from L in the compounds of formula I. Preferably Fcomprises a succinimidyl ester group, phosphoramidite group, maleimidegroup, biotin or azide group.

Preferably F is or comprises a phosphoramidite group. The functionalisedderivative may therefore be a compound according to formula IIA:

-   -   wherein:        -   each X substituent is independently selected from halo,            vinyl, alkyl, cycloalkyl, SiR₃, SnR₃, PR₂, P(O)R₂, SR,            S(O)R, SO₂R, aryl, heteroaryl, CHO, CO₂R, CN and CF₃;        -   each R is independently selected from alkyl, cycloalkyl,            aryl and heteroaryl;        -   A is O, B is CH₂ and c is 1, or        -   A is CH₂, B is O and c is 2;        -   R′ is alkyl;        -   R″ is alkyl;        -   a is 0, 1, 2, 3 or 4;        -   b is 0, 1, 2, 3, 4 or 5; and    -   vinyl, alkyl, cycloalkyl, alkylene, aryl and heteroaryl may        optionally be substituted with 1, 2 or 3 substituents        independently selected from unsubstituted alkyl, OH, CN,        fluorine, chlorine, bromine and iodine.

Preferably R′ is i-propyl and R″ is —CH₂CH₂CN. Compounds of formula IIAmay be formed by functionalisation of a compound of formula IA with afunctionalising compound comprising a phosphoramidite group.Functionalisation with phosphoramidite is particularly advantageous forattaching compounds of the invention to nucleic acids. The linking ofphosphoramidite groups to nucleic acids is well-known and a routinematter to those skilled in the art.

Labelled substrates according to the invention may be prepared byreaction of a compound or functionalised derivative of the invention,with a substrate. Thus, a labelled substrate may be of formula

-   -   wherein:        -   each X substituent is independently selected from halo,            vinyl, alkyl, cycloalkyl, SiR₃, SnR₃, PR₂, P(O)R₂, SR,            S(O)R, SO₂R, aryl, heteroaryl, CHO, CO₂R, CN and CF₃;        -   each R is independently selected from alkyl, cycloalkyl,            aryl and heteroaryl;        -   Y is a spacer;        -   Z is a spacer;        -   L′ is the residue of L or F as described above, after a            compound or functionalised derivative of the invention is            reacted with a substrate;        -   a is 0, 1, 2, 3 or 4;        -   b is 0, 1, 2, 3, 4 or 5;        -   [S] is the residue of a substrate; and    -   vinyl, alkyl, cycloalkyl, alkylene, aryl and heteroaryl may        optionally be substituted with 1, 2 or 3 substituents        independently selected from unsubstituted alkyl, OH, CN,        fluorine, chlorine, bromine and iodine.

L′ may be the residue of L or F as described above, after a compound orfunctionalised derivative of the invention is reacted with a substrate.Preferably L′ is the residue of a hydroxy group or a phosphoramiditegroup. In an embodiment [S] is not the residue of a single nucleotide.In an embodiment, [S] is the residue of a polypeptide, amino acid orcarbohydrate.

Illustrative compounds of the invention are shown in Table 1 below.

TABLE 1 Illustrative compounds of the invention

  3-(ferrocenylmethoxy)propan-1ol (1)

  3-(nonamethylferrocenylmethoxy)propan-1-ol (2)

  3-((1′-chloro)-ferrocenylmethoxy)propan- 1-ol (3)

  3-((2-tert-butylthio)-ferrocenylmethoxy)propan-1-ol (4)

  3-((2-tert-butylsulfinyl)- ferrocenylmethoxy)propan-1-ol (5)

  3-((2-tert-butylsulfonyl)-ferrocenylmethoxy)propan-1-ol (6)

  3-((2-di-tert-butylphospinyl)- ferrocenylmethoxy)propan-1-ol (7)

  3-(2-tributylstannyl-ferrocenylmethoxy)propan-1-ol (8)

  2-trimethylsilyl- ferrocenylmethoxy)propan-1-ol (9)

  3-(2-tributylsilyl-ferrocenylmethoxy)propan-1-ol (10)

  3-(2-trimethylstannyl- ferrocenylmethoxy)propan-1-ol (11)

  3-(2-Vinyl-ferrocenylmethoxy)propan-1-ol (12)

  3-(2-iodo-ferrocenylmethoxy)propan-1-ol (13)

  2-(3-ferrocenylpropoxy)ethanol (14)

  2-(3-(2-tert-butylthio)- ferrocenylpropoxy)ethanol (15)

  2-(3-(2-tert-butylsulfinyl)-ferrocenylpropoxy)ethanol (16)

  2-(3-(2-tert-butylsulfonyl)- ferrocenylpropoxy)ethanol (17)

Any of the compounds in Table 1 may be functionalised by any suitablemethod, for example by phosphoramidation. Illustrative functionalisedcompounds of the invention, functionalised with a phosphoramidite group,are shown in Table 2 below. The present invention encompasses labelledsubstrates derived from the compounds in Table 1 and 2.

TABLE 2 Illustrative functionalised compounds of the invention

  2-cyanoethyl-(2(3- ferrocenylpropoxy)ethanol)di-iso-propyl-phosphoramidite

  2-Cyanoethyl-(3- (Nonamethylferrocenylmethoxy)propan-1-ol)di-iso-propylphosphoramidite

It is believed that compounds of the invention, particularly thosehaving sulfur-containing or phosphorus-containing substituents on theferrocenyl moiety, and their corresponding functionalised derivativesand labelled substrates, will be useful in assays in which themeasurement potential will be relatively high, for example, in excess of400 mV, for example in excess of 450 mV or even in excess of 500 mV.Compounds having electrochemical potentials of at least 450 mV, forexample 500 mV or more, will be particularly useful in extending therange of available potential values and therefore, for example, inpotentially providing for more effective multiplex assays. Compounds ofthe invention having highly electron-withdrawing substituents on theferrocenyl moiety, for example, trifluoromethyl or cyano, are believedto have similar advantages in terms of offering high electrochemicalpotential values thereby extending the range of useful labels andlabelled substrates. Compounds of the invention that are electron rich,such as compound 2, are useful for extending the range ofelectrochemical potentials to low voltages, for example to a voltage <0mV. This is particularly advantageous for extending the scope ofmultiplex assays.

Additionally, some compounds of the invention, particularly thosecompounds bearing halogen atoms, and the corresponding labelledsubstrates offer the advantage of having a narrower voltage peak, whichis advantageous in providing for the option of utilising a greaternumber of labels in a multiplex assay, since the narrower measurementpeaks result in wider gaps between peaks, which may be utilised ifdesired by incorporating additional labels with potentials that will bewithin the gaps.

Electrochemical detection is based on the observation that anelectrochemically active marker exhibits different electrochemicalcharacteristics depending on whether or not it is attached to asubstrate and on the nature of the substrate. For example, in the caseof an electrochemical label attached to an amino acid, the exhibitedcharacteristics will depend not only on the identity of the amino acidbut also on whether or not that amino acid residue is incorporated intoa polypeptide, and on the length of any such polypeptide. Underappropriate circumstances, the electrochemical activity of a markerattached to an amino acid residue can change by a detectable degreefollowing loss of attachment of a single or very few amino acidresidues.

The size and characteristics of a substrate to which anelectrochemically active marker is attached influence the observablecharacteristics of the electrochemical marker. Without wishing to bebound by theory, such a change in the observable characteristics of theelectrochemical may occur, for example, by influencing the rate ofmigration of the marker by diffusion or its rate of migration inresponse to an electric field.

Electrochemical activity of a marker may also be influenced by stericeffects resulting from the presence of the molecule to which it islinked. For example, steric hindrance may prevent the marker fromapproaching an electrode and accepting or donating electrons.

If the marker is attached to a polypeptide then the secondary structureof the polypeptide (as largely determined by the primary sequence) mayinfluence the physical properties of the marker. For example, if themarker is attached to an amino acid residue in a polypeptide such thatthe structure of the polypeptide sterically hinders theelectrochemically active marker then the signals observable byvoltammetry may be reduced. Digestion of the polypeptide may destroy orrelease secondary structure elements and thus reduce or abolish theinfluence of the peptide structure on the marker. Accordingly, digestionof the polypeptide results in a change, usually an increase, in theelectrochemical signal produced by the marker moiety. In a differentialpulse voltammetry experiment, the Faradaic current response at aparticular applied voltage may increase upon digestion of the peptide.

The information relating to the electrochemically active marker can beobtained by voltammetry or by an amperometric method. Differential pulsevoltammetry is particularly suitable. If desired, the electrochemicaldetection step may be carried out using one or more electrodes coveredby a membrane which is able to selectively exclude molecules based onone or more characteristics, for example, size, charge orhydrophobicity. That may assist in eliminating background noise currentarising from, for example, charged species in the solution.

Analogously, if a marker is attached to a nucleotide, theelectrochemical characteristics will be influenced by whether or not thenucleotide is incorporated into a more complex nucleic acid such as apolynucleotide, upon the length of that nucleic acid, and upon thesequence of the nucleic acid, especially in the vicinity of the point ofattachment.

The invention also provides a method of detecting a nucleic acid (forexample RNA or DNA) in a sample comprising the optional step ofamplifying the nucleic acid (for example by PCR or another nucleic acidamplification technique) followed by the step of contacting the amplicon(or the nucleic acid) with a complementary nucleic acid probe underconditions to allow hybridization between the probe and amplicon (or thenucleic acid), followed by the step of selectively degrading eitherhybridised or unhybridised probe (for example by use of single or doublestrand specific nucleases), wherein said probe is labelled with anelectrochemically active compound of the invention and wherein themethod provides the step of measuring the electrochemical activity ofthe compound labelling the probe of wherein said electrochemicalactivity is dependent either quantitatively or qualitatively on theextent of degradation of the probe. Such use of electrochemical labelsin nucleic acid hybridisation assays is described by Pearce et al.(2011) IEEE Trans Biomed Eng 58:755-58, the complete contents of whichare incorporated herein by reference.

The invention also provides a method of detecting an antibody orderivative (which may for example be bound to target antigen in anassay) with an electrochemically active compound of the inventioncomprising the step of measuring the electrochemical activity of thecompound. This method can be performed quantitatively or qualitatively.

The invention also provides methods of diagnosing or monitoring adisease in a subject comprising using a method of the invention in thedetection of a protease or a protease inhibitor associated with saiddisease in a tissue or body fluid of the subject. A substrate for theprotease can be labelled according to the invention. Examples of diseasethat are associated with the presence of a protease or a proteaseinhibitor in a tissue of the subject include hereditary predispositionto thromoembolism caused to deficiencies in anti-thrombin III in theblood serum. Elevated serum or extracellular matrix cathepsin levels maybe indicative of Alzheimer's disease, cancer or arthritis. Preferablythe tissue or body fluid of the subject is serum, plasma, saliva, urineor any other tissue or body fluid of which a sample may be convenientlyand safely obtained.

The invention also provides methods of diagnosing a disease in a subjectcomprising using a method of the invention to detect a polypeptideassociated with said disease in a tissue or body fluid of the subject.

The invention also provides methods of diagnosing or monitoring adisease in a subject comprising using a method of the invention in thedetection of a nuclease or a nuclease inhibitor associated with saiddisease in a tissue or body fluid of the subject.

Furthermore, the invention provides use of a method of the invention fordetecting a disease in a subject. The invention also provides methods ofdetecting a microorganism (in particular, a pathogen or otherundesirable organism, for example a food spoilage organism), comprisingusing a method of the invention. A substrate from the microorganism (orderived from the pathogen e.g. a nucleic acid amplicon produced using atarget nucleic acid sequence in the pathogen) can be labelled accordingto the invention. Detection of the labelled substrate can be used toindicate detection of the microorganism. Preferably the microorganism isselected from the group consisting of Chlamydia trachomatis, Trichomonasvaginalis, Neisseria gonorrhoeae, Mycoplasma genitalium and methicillinresistant Staphylococcus aureus.

The invention also provides an assay comprising a step which uses alabelled substrate of the invention, optionally in combination withother assay components for example a sample vessel, a containercomprising electrodes for electrochemical detection, enzymes for use inthe assay or standards and controls. Said assay may use more than onedifferent labelled substrate of the invention. If that is the case thepresence of different labelled substrates may be differentially detectedby labelling them with electrochemical labels of the invention havingdifferent electrochemical characteristics (for example differentoxidation potentials) thereby permitting the assay to be a multiplex(for example a duplex) assay in which different substrates may bediscriminated when present in the same sample vessel. Simplex assays arealso encompassed by the invention.

As illustrated in the examples, incorporation of one or moresubstituents on the ferrocenyl groups can be used to obtain compoundswith modified electrochemical characteristics to be used in assays.Moreover, the invention provides a range of compounds from which two ormore may be selected for use in multiplex reactions and assays.

Attachment of a compound or a functionalised derivative of the inventionto a substrate can be by any suitable linkage, typically by linkage to asubstrate side chain. Conventional hydroxy protecting groups, forexample those described by T. W. Greene and P. G. M. Wuts in “Protectivegroups in organic chemistry” John Wiley and Sons, 4th Edition, 2006, maybe used. A common hydroxy protecting group suitable for use herein is amethyl ether; deprotection conditions can comprise refluxing in 48%aqueous HBr for 1-24 hours, or by stirring with borane tribromide indichloromethane for 1-24 hours. Alternatively a hydroxy group may beprotected as a benzyl ether; deprotection conditions can comprisehydrogenation with a palladium catalyst under a hydrogen atmosphere.

Various synthetic methods are known in the art for the derivatisation ofsubstrates. For example, lysine or lysine residues may be derivatised byreaction with a succinimidyl ester. For derivatisation of other aminoacids and amino acid residues, other known synthetic methods may beused. For example, a maleimide reagent may be used to derivatisecysteine or cysteine residues. An N-hydroxysuccinimide ester may be usedto derivatise the amino terminus or side chain amino group of apolypeptide or an amino acid. Suitable derivatisation methods fornucleic acids are also well-known, for example, using a phosphoramiditemoiety.

A compound of the invention may be attached to a substrate by use of anyfunctionalising group that facilitates attachment of a labellingcompound to a substrate. Suitable functionalising groups includesuccinimidyl ester groups, phosphoramidite groups, maleimide groups,biotin and azide groups.

Attachment of a compound of the invention to a polypeptide, for examplevia cysteine or lysine, may be accomplished in some cases by incubationof the polypeptide and compound of the invention together at roomtemperature in an appropriate buffer solution. Where the label isadvantageously to be linked to cysteine or lysine but the substratesequence does not contain cysteine or lysine at a suitable position thesequence may, if desired, be mutated to add one or more cysteine orlysine residue either as an additional residue or as a substitution foranother residue. An alternative method for attachment to polypeptidesincludes biotinylation of the labels and use of commercialstreptavidinated proteins (or vice versa). By way of example, thesubstrate may be biotinylated by any standard technique for example byuse of a commercially available biotinylation kit. Biotinylatedsubstrate will bind to strepavidin or avidin conjugated compounds suchas antibodies, which are commercially and widely available.

In an embodiment, the compound of formula I is not

In an embodiment, the compound of formula I is not

In an embodiment the invention does not include

In an embodiment the compound of formula I is not any of

In an embodiment of the compounds of the invention, Z is not —CH₂CH₂—.In an embodiment of the compounds of the invention, Y is not —CH(Me)-.In an embodiment X is not PPh₂.

EXAMPLES

Compounds of the invention can be prepared according to the proceduresof the following schemes and examples, using appropriate materials.Moreover, by utilising the procedures described herein, one of ordinaryskill in the art can readily prepare additional compounds that fallwithin the scope of the present invention. The reader will readilyunderstand that known variations of the conditions and processes of thefollowing preparative procedures can be used to prepare these compounds.Thus, the invention is not to be construed as being limited to thecompounds illustrated in the examples.

The following abbreviations have been used in the examples:

DMSO Dimethylsulfoxide THF Tetrahydrofuran DIPEAN,N-diisopropylethylamine PCR polymerase chain reaction pTSAp-toluenesulfonic acid Tf trifluoromethanesulfonate eq equivalent(s) TLCthin layer chromatography sat saturated HRMS high resolution massspectrometry ESI electrospray ionisation

Compounds according to general formula I and II may be prepared usingconventional synthetic methods, for example, but not limited to, theroutes outlined in the schemes below. More detailed synthetic procedurescan be found in the examples below.

E⁺ is any suitable electrophile useful for substituting a ferrocenylgroup.

Scheme 3 illustrates the general synthetic procedure for attaching aphosphoramidite functional group to a linker hydroxyl group.

The ferrocenyl derivative shown as a starting material in the abovereaction scheme is illustrative, and may be replaced by a molarequivalent of any of the compounds of the invention.

Determination of Electrochemical Potential

The electrochemical potential values mentioned hereafter were measuredusing an electrochemical cell including as background electrolyte anaqueous 100 mM solution of sodium chloride, using a printed carbonworking electrode, a printed carbon counter electrode and asilver/silver chloride reference electrode, all with silver connectors.The electrodes were ink based and were screen printed on to a polymersubstrate (for example Mylar®) followed by heat curing. By way ofillustration, the sample may be prepared as follows: 0.01 M stocksolution of the ferrocenyl compound is prepared in DMSO (1 cm³). This isthen further diluted to 14 μM in buffer. A 20 μL aliquot of this 14 μMsolution is then applied to the screen printed electrode to run theelectrochemical scan. An illustrative form of suitable cell is describedand shown schematically in WO2012/085591.

Example 1: Preparation of 3-(ferrocenylmethoxy)propan-1ol (1)

To a round bottomed flask equipped with a magnetic stirrer bar was addedferrocene carboxaldehyde (535 mg, 2.5 mmol, 1 eq). The flask was thencharged with ethanol (4 cm³) and THF (1 cm³). The red solution was thentreated with sodium borohydride (123 mg, 3.2 mmol, 1.3 eq). The flaskwas then sealed and placed under a nitrogen atmosphere. After 30 minutesthe solution had changed colour to an orange and TLC analysis indicatedfull consumption of the starting material. The flask was thenconcentrated to ˜90% of original volume in vacuo. The dark orangeresidue was then taken up in EtOAc (15 cm³) and NaHCO₃ (15 cm³). Thebi-phasic mixture was transferred to separating funnel, the aqueouslayer was separated and then back extracted with EtOAc (3×5 cm³), thecombined organic washings were then dried over MgSO₄, filtered and thenconcentrated in vacuo to give a yellow solid. The ferrocene methanol wasthen taken up in 1,3-propanediol (5 cm³), the yellow solution was thentreated with ytterbium (III) triflate (77 mg, 0.125 mmol, 5 mol %). Theflask was then sealed and heated to 100° C. After heating for 10 minutesTLC analysis indicated full consumption of the starting material. Theflask was cooled to room temperature, diluted with H₂O (20 cm³) andEtOAc (20 cm³). The organic layer was then separated and the aqueouslayer back extracted with EtOAc (3×5 cm³). The combined organic layerswere then washed with H₂O (20 cm³) and brine (sat) (20 cm³) then driedover MgSO₄, filtered then concentrated in vacuo to give an orange solid.Purification was then carried out by silica-gel chromatography elutingwith n-Hex 1:1 EtOAc to give the desired product3-(ferrocenylmethoxy)propan-1ol (1) as an orange powder (514 mg, 74%).

¹H NMR (250 MHz, CDCl₃); δ_(H): 4.24 (s, 4H), 4.11 (s, 6H), 3.65 (t, 2H,J=5.4 Hz), 3.54 (t, 2H J=5.4 Hz), 3.65 (t, 2H J=5.4 Hz), 2.52 (br s,1H), 1.7 (quin 2H, J=5.6 Hz); ¹³C NMR (75 MHz, CDCl₃); δ_(C): 83.6,77.3, 71.5, 69.4, 69.3, 69.2, 68.7, 32.0; HRMS (ESI μTOF) calculated forC₁₄H₁₈FeO₂Na m/z 297.0553 found 297.0560 (m/z+Na⁺); Electrochemicalpotential: 181 mV.

Example 2: Preparation of 3-(nonamethylferrocenylmethoxy)propan-1-ol (2)

Nonamethylferrocene carboxaldehyde (B)

Decamethylferrocene (A) (4.80 g, 14.7 mmol) was placed in a roundbottomed flask equipped with a magnetic stirrer bar. Fresh finely groundbarium manganate (18.77 g, 73.6 mmol, 5 eq) was then added to the flask.The solids were then suspended in a mixture of dry benzene (20 cm³) anddry diethyl ether (20 cm³). The flask was then sealed and placed under anitrogen atmosphere. The dark blue slurry was then sonicated for 45mins. After this time the flask was removed from the sonicater andheated at 45° C. for 16 hours. After this time the dark slurry wasfiltered through a pad of celite and the solids washed with EtOAc (250cm³) until the washings ran clear. The red solution was thenconcentrated in vacuo to give a red solid. Purification by silicachromatography eluting with 5% EtOAc:nHex+2% TEA gave the productnonamethylferrocene carboxaldehyde (B) as a dark red crystalline solid(1.19 g, 23%).

¹H NMR (300 MHz, CDCl₃) δ_(H): 9.91 (s, 1H), 1.92 (s, 6H), 1.71 (s, 6H),1.59 (s, 15H). ¹³C NMR (75 MHz, CDCl₃) δ_(C): 195.6, 86.0, 82.7, 80.6,78.3, 72.5, 9.3, 9.3, 8.9. HRMS (ESI μTOF) calculated for C₂₀H₂₉FeO m/z341.1484 found 341.1485 (m/z+H).

3-(Nonamethylferrocenylmethoxy)propan-1-ol (2)

The nonamethylferrocene carboxaldehyde (B) (3.43 g, 10.08 mmol, 1 eq)was placed in a round bottomed flask equipped with a magnetic stirrerbar. The flask then charged with ethanol (44 cm³) and 1,4-dioxane (11cm³), the red solution was then treated with sodium borohydride (820 mg,22.18 mmol, 2.2 eq). The flask was then sealed, placed under an argonatmosphere and stirred at room temperature for 16 hours. After this timeTLC analysis indicated full consumption of the starting material. Theorange solution was concentrated in vacuo to approximately 90% oforiginal volume. The orange solid was then partitioned between H₂O (50cm³) and CH₂Cl₂ (50 cm³). The organic layer was separated and theaqueous layer was back extracted with CH₂Cl₂ (3×15 cm³). The combinedorganics were then combined, washed with brine (sat) (50 cm³), driedover MgSO₄, filtered and concentrated in vacuo to give an orange solid.The crude alcohol was then suspended in 1,3-propanediol (50 cm³), CH₂Cl₂(10 cm³) to give a red solution. The solution was then treated withytterbium (III) triflate (334 mg, 0.54 mmol, 5 mol %). The flask wasthen sealed and placed under nitrogen atmosphere. After stirring for 30mins at room temperature TLC analysis showed full consumption of thestarting material. The reaction was then diluted with H₂O (150 cm³) andCH₂Cl₂ (100 cm³). The organic layer was separated and the aqueous layerextracted with CH₂Cl₂ (3×15 cm³). The combined organics were then washedwith H₂O (3×50 cm³), dried over MgSO₄, filtered and concentrated invacuo to give an orange oil. Purification by silica chromatographyeluting with 10% EtOAc:nHex+2% TEA to give the desired product3-(Nonamethylferrocenylmethoxy)propan-1-ol (2) as a yellow powder 3.16g, 78%.

¹H NMR (300 MHz, C₆D₆) δ_(H): 4.31 (s, 2H), 3.71 (s, 2H), 3.52 (t, 2H,J=5.7 Hz), 2.18 (s, 2H), 1.85 (s, 6H), 1.70 (s, 22H); ¹³C NMR (75 MHz,CDCl₃); δ_(C): 83.6, 77.3, 71.5, 69.4, 69.3, 69.2, 68.7, 32.0; HRMS (ESIμTOF) calculated for C₂₃H₃₆FeO₂Na m/z 423.1962 found 423.1955 (m/z+Na⁺);Electrochemical potential: −151 mV.

Example 3: Preparation of 3-((1′-chloro)-ferrocenylmethoxy)propan-1-ol(3)

Chloroferrocene (C) was prepared from ferrocene using a modifiedprocedure from J. Organomet. Chem., 1996, 512, 219-224, usinghexachloroethane as chlorinating reagent.

1′-Chloroferrocenecarboxaldehyde (D) was prepared from chloroferrocene,using the procedure from Coll. Chechoslovak. Chemm. Commun., 1987, 52,174-181, as a 4:1 mixture of the desired regioisomer.

The 1′-chloroferrocenecarboxaldehyde (D) (426 mg, 1.7 mmol, 1 eq) wasplaced in a round bottomed flask equipped with magnetic stirrer bar anddissolved in ethanol (4 cm³) and THF (1 cm³). The red solution was thentreated with sodium borohydride (83 mg, 2.2 mmol, 1.3 eq), the flask wassealed and placed under a nitrogen atmosphere. After 30 mins thesolution had turned orange in colour and TLC analysis indicated fullconsumption of the starting material. The flask was then concentrated to˜90% of original volume in vacuo. The dark orange residue was then takenup in EtOAc (15 cm³) and NaHCO₃ (sat) (15 cm³). The bi-phasic mixturewas transferred to a separating funnel, the aqueous layer was separatedand then back extracted with EtOAc (3×5 cm³). The combined organicwashings were then combined dried over MgSO₄, filtered and thenconcentrated in vacuo to give an orange/yellow oil. The ferrocenemethanol was then taken up in 1,3-propanediol (3 cm³), the yellowsolution was then treated with ytterbium (III) triflate (56 mg, 0.09mmol, 5 mol %). The flask was then sealed and heated to 100° C., afterheated for 10 minutes TLC analysis indicated full consumption of the thestarting material. The flask was cooled to room temperature, dilutedwith H₂O (10 cm³) and EtOAc (10 cm³). The organic layer was thenseparated and the aqueous layer back extracted with EtOAc (3×5 cm³). Thecombined organic layers were then washed with H₂O (20 cm³) and brine(sat) (20 cm³), dried over MgSO₄, filtered, then concentrated in vacuoto give a brown oil. Purification was then carried out by silica-gelchromatography eluting with 25% EtOAc:nHex to give the desired product3-((1′-chloro)-ferrocenylmethoxy)propan-1-ol (3) as an orange oil (297mg, 57%) as a 4:1 mixture of regioisomers.

¹H NMR (300 MHz, C₆D₆) δ_(H)(major) 4.26 (t, J=1.9 Hz, 2H), 4.19-4.13(m, 4H), 4.03 (t, J=1.9 Hz, 2H), 3.78 (t, J=1.9 Hz, 2H), 3.69 (s, 2H),3.46 (t, J=5.8 Hz, 2H), 1.73-1.65 (m, 2H); ¹³C NMR (75 MHz, C₆D₆) δ_(C)(major) 93.3, 86.2, 71.5, 71.0, 69.6, 69.3, 69.0, 67.2, 62.0, 33.1; HRMS(ESI μTOF) calculated for C₁₄H₁₇ClFeO₂Na m/z 331.0164 found 331.0144(m/z+Na⁺); Electrochemical potential: 352 mV.

Example 4: Preparation of3-((2-tert-butylthio)-ferrocenylmethoxy)propan-1-ol (4)

1-[(Dimethylamino)methyl]-2-(t-butylthio)-ferrocene (E) was preparedusing the procedure from Organomet., 1988, 7, 1297-1302.

1-[(Acetoxy)methyl]-2-(tert-butylthio)-ferrocene (F)

1-[(Dimethylamino)methyl]-2-(t-butylthio)-ferrocene (E) (1.21 g, 3.49mmol) was dissolved in acetic anhydride (10 cm³). The brown solution wasthen refluxed for 1 hour; TLC at this time indicated full consumption ofthe starting material. The solution was allowed to cool to roomtemperature, the solution was then concentrated in vacuo toapproximately 90% of original volume. The resulting brown oil was thentaken up in EtOAc (25 cm³) and washed with NaHCO₃ (sat) (20 cm³) andbrine (sat) (20 cm³). The brown solution was then dried over MgSO₄,filtered and concentrated in vacuo to give1-[(acetoxy)methyl]-2-(tert-butylthio)-ferrocene (F) as an orange/brownoil (1.12 g, 93%) without need for further purification.

¹H NMR (250 MHz, C₆D₆) δ_(H) 5.37 (d, J=1.43 Hz, 2H), 4.51 (dd, J=2.6,1.4 Hz, 1H), 4.44 (dd, J=2.6, 1.4 Hz, 1H), 4.07 (t, J=2.6 Hz, 1H), 4.07(s, 5H) 1.82 (s, 3H), 1.33 (s, 9H).

2-tert-butylthio Ferrocene Methanol (G)

To a suspension of lithium aluminium hydride (369 mg, 9.71 mmol) in Et₂O(15 cm³) at 0° C. was added1-[(Acetoxy)methyl]-2-(tert-butylthio)-ferrocene (F) (1.12 g, 3.23 mmol)dropwise via syringe. Once addition was complete the slurry was allowedto warm to room temperature and stir for 30 mins. After this time theflask was cooled to 0° C. and then quenched by sequential addition ofH₂O (369 μl), followed by 15% NaOH (aq) (369 μl) and H₂O (1.1 cm³). Thesuspension was then allowed to warm to room temperature stirred for 10minutes, filtered and concentrated in vacuo to give 2-tert-butylthioferrocene methanol (G) as an orange solid (790 mg, 80%) without the needfor further purification.

¹H NMR (250 MHz, C₆D₆) δ_(H) 4.64 (s, 2H), 4.41 (dd, J=2.4, 1.5 Hz, 1H),4.32 (dd, J=2.4, 1.5 Hz, 1H), 4.17 (s, 5H), 4.08 (t, J=2.6 Hz, 1H), 1.28(s, 9H). 3-((2-tert-butylthio)-ferrocenylmethoxy)propan-1-ol (4).

The 2-tert-butylthio ferrocene methanol (G) (778 mg, 2.5 mmol) wasplaced in a round bottomed flask equipped with a magnetic stirrer bar,and then suspended in 1,3-propandiol (10 cm³). The yellow suspension wasthen treated with ytterbium (III) triflate (79 mg, 0.125 mmol, 5 mol %),the flask sealed, placed under a nitrogen atmosphere then heated to 100°C. After 10 mins TLC analysis indicated full consumption of the startingmaterial. The brown solution was allowed to cool to room temperature,then diluted with H₂O (20 cm³) and EtOAc (20 cm³). The organic layer wasseparated and the aqueous layer back extracted with EtOAc (3×10 cm³).The combined organics were washed with brine (sat) (2×10 cm³), driedover MgSO₄, filtered and concentrated in vacuo to give an orange oil.Purification by silica chromatography eluting with 20% EtOAc:n-Hex togive the desired product3-((2-tert-butylthio)-ferrocenylmethoxy)propan-1-ol (4) as an orange oil(899 mg, 99%).

¹H NMR (300 MHz, C₆D₆) δ_(H) 4.55 (d, J=10.7 Hz, 1H), 4.42 (dd, J=2.5,1.4 Hz, 1H), 4.40 (dd, J=2.6, 1.6, 1H), 4.32 (d, J=10.7 Hz, 1H), 4.14(s, 5H), 4.09 (t, J=2.6 Hz, 1H), 3.73 (q, J=5.8 Hz, 2H), 3.57 (qt,J=9.0, 5.8 Hz, 2H), 2.10 (t, J=5.8 Hz, 1H), 1.74 (p, J=5.8 Hz, 2H), 1.32(s, 9H). ¹³C NMR (75 MHz, C₆D₆) δ_(C) 88.6, 77.7, 77.5, 71.0, 70.7,70.2, 69.5, 68.4, 62.0, 45.7, 33.2, 31.4; HRMS (ESI μTOF) calculated forC₁₈H₂₆FeO₂SNa m/z 385.0918 found 385.0900 (m/z+Na⁺); Electrochemicalpotential: 352 mV.

Example 5: Preparation of3-((2-tert-butylsulfinyl)-ferrocenylmethoxy)propan-1-ol (5)

The 3-((2-tert-butylthio)-ferrocenylmethoxy)propan-1-ol (4) (459 mg, 1.2mmol, 1 eq) was dissolved in CH₂Cl₂ (10 cm³), the flask was then placedunder a nitrogen atmosphere and cooled to 0° C. Once cold3-chloro-perbenzoic acid (258 mg, 1.5 mmol, 1.2 eq) was added in oneportion. The solution was then stirred at 0° C. for 15 minutes. Afterthis time TLC analysis indicated full consumption of the startingmaterial. The reaction was then quenched by addition of NaHCO₃ (sat) (15cm³) and stirred vigorously for 5 minutes. After this time the organiclayer was separated and aqueous layer extracted with CH₂Cl₂ (3×5 cm³).The combined organic were then washed with brine (sat) (10 cm³), driedover MgSO₄, filter and concentrated in vacuo to give a dark brown oil.Purification by silica chromatography eluting with EtOAc gave thedesired product 3-((2-tert-butylsulfinyl)-ferrocenylmethoxy)propan-1-ol(5) as orange amorphous solid (349 mg, 77%).

¹H NMR (300 MHz, C₆D₆) δ_(H) 4.82 (dd, J=2.6, 1.5 Hz, 1H), 4.38 (s, 5H),4.34-4.22 (m, 2H), 4.18 (d, J=11.1 Hz, 1H), 4.11 (t, J=2.6 Hz, 1H), 3.77(q, J=5.5 Hz, 2H), 3.55 (ddd, J=9.0, 5.5, 5.5, Hz, 1H), 3.46 (ddd,J=9.0, 5.5, 5.5 Hz, 1H), 2.59 (t, J=5.5 Hz, 1H), 1.79 (p, J=5.5 Hz, 2H),1.19 (s, 8H); ¹³C NMR (75 MHz, C₆D₆) δ_(C) 88.7, 87.1, 72.0, 71.4, 69.7,69.4, 68.0, 66.9, 61.1, 55.8, 33.4, 23.4; HRMS (ESI μTOF) calculated forC₁₈H₂₆FeO₃SNa m/z 401.08497 found 401.0838 (m/z+Na⁺); Electrochemicalpotential: 474 mV.

Example 6: Preparation of3-((2-tert-butylsulfonyl)-ferrocenylmethoxy)propan-1-ol (6)

The 3-((2-tert-butylsulfinyl)-ferrocenylmethoxy)propan-1-ol (5) (349 mg,0.92 mmol, 1 eq) was dissolved in CH₂Cl₂ (10 cm³), the flask was thenplaced under a nitrogen atmosphere and cooled to 0° C. Once cold,3-chloro-perbenzoic acid (190 mg, 1.1 mmol, 1.2 eq) was added in oneportion. The solution was then stirred at 0° C. for 30 minutes. Afterthis time TLC analysis indicated full consumption of the startingmaterial. The reaction was then quenched by addition of NaHCO₃ (sat) (15cm³) and stirred vigorously for 5 minutes. After this time the organiclayer was separated and aqueous layer extracted with CH₂Cl₂ (3×5 cm³).The combined organic were then washed with brine (sat) (10 cm³), driedover MgSO₄, filter and concentrated in vacuo to give a dark brown oil.Purification by silica chromatography eluting with 40% EtOAc:n-Hex gavethe desired product3-((2-tert-butylsulfonyl)-ferrocenylmethoxy)propan-1-ol (6) as a yellowsolid (256 mg, 70%).

¹H NMR (300 MHz, C₆D₆) δ_(H) 4.77 (d, J=11.0 Hz, 1H), 4.58 (dd, J=2.5,1.6 Hz, 1H), 4.46 (d, J=11.0 Hz, 1H), 4.40 (s, 5H), 4.36 (dd, J=2.5, 1.6Hz, 1H), 4.03 (t, J=2.5 Hz, 1H), 3.71 (t, J=5.8 Hz, 2H), 3.61-3.50 (m,2H), 2.05 (s, 1H), 1.77-1.68 (m, 2H), 1.27 (s, 9H); ¹³C NMR (75 MHz,C₆D₆) δ_(C) 86.7, 82.9, 73.7, 73.2, 72.1, 70.4, 70.1, 67.5, 61.7, 59.2,33.2, 23.9; HRMS (ESI μTOF) calculated for C₁₈H₂₇FeO₄SNa m/z 418.0833found 418.0824 (m/z+H); Electrochemical potential: 584 mV.

Example 7: Preparation of3-((2-di-tert-butylphosphinyl)-ferrocenylmethoxy)propan-1-ol (7)

The 1-[(dimethylamino)methyl]-2-(t-di-tert-butylphoshonyl)-ferrocene (H)(4.82 g, 12.5 mmol, 1 eq) was placed in a round bottomed flask with amagnetic stirrer bar, and dissolved in acetone (30 cm³).

The orange solution was then cooled to 0° C. Once cold the solution wastreated with H₂O₂ (50% wt) (812 μl, 14.3 mmol, 1.15 eq) dropwise over a2 minute period. Once addition was complete the flask was allowed towarm to room temperature, after stirring for 15 minutes the reaction wascomplete. The flask was re-cooled to 0° C. and then quenched by additionof Na₂S₂O₃ (sat) (30 cm³). The solution was further diluted with EtOAc(50 cm³), the organic layer was separated and the aqueous layer backextracted with EtOAc (3×30 cm³). The combined organics were then washedwith brine (sat) (30 cm³), dried over MgSO₄, filtered and concentratedin vacuo to give a thick red oil. This red oil was taken up in aceticanhydride (30 cm³), then heated at 100° C. for 2 hours. After this timethe solution was allowed to cool to room temperature, the brown solutionwas then concentrated to approximately 90% of original volume. Theresulting brown oil was then taken up in EtOAc (50 cm³). The solutionwas then washed with 2M NaOH (20 cm³), H₂O (2×20 cm³) and brine (sat)(50 cm³). The organic layer was then dried over MgSO₄, filtered and thenconcentrated in vacuo to give a brown oil. Purification by basic aluminachromatography, eluting with EtOAc gave the desired product1-[(acetoxy)methyl]-2-(di-tert-butylphosphinyl)-ferrocene (I) as a redoil (2.30 g, 44%). ¹H NMR (300 MHz, C₆D₆) δ_(H) 5.87 (d, J=12.3 Hz, 1H),5.62 (d, J=12.3 Hz, 1H), 4.58-4.47 (m, 1H), 4.19-4.06 (m, 6H), 3.80(brs, 1H), 1.85 (s, 3H), 1.48 (d, J^(P-H)=16.7 Hz, 9H), 1.21 (d,J^(P-H)=16.7 Hz, 9H); ³¹P{H} NMR (122 MHz, C₆D₆) δ_(P) 58.59; ¹³C NMR(75 MHz, C₆D₆) δ_(C) 170.5, 72.5, 71.3, 62.4, 60.4, 42.2, 41.4, 38.0,37.3, 37.2, 36.5, 27.7, 27.0, 26.9, 21.1, 14.6; HRMS (ESI μTOF)calculated for C₂₁H₃₁FeO₃PNa m/z 441.1257 found 441.1265 (m/z+Na⁺).

2-(di-tert-Butyl-phosphinyl)-ferrocene Methanol (J)

To a suspension of lithium aluminium hydride (628 mg, 16.5 mmol, 3 eq)in dry diethyl ether (10 cm³) under nitrogen at 0° C., was added the2-di-tert-butylphosphinyl-acetoxy methyl ferrocene (I) (2.30 g, 5.5mmol, 1 eq) in dry diethyl ether (10 cm³) dropwise via syringe over a 2minute period. Once addition was complete the flask was then refluxedovernight. After this time the flask was then cooled to 0° C. and thereaction was then quenched by sequential addition of H₂O (628 μl), 15%NaOH (aq) (628 μl) and H₂O (1.88 cm³). The orange slurry was thenallowed to stir at room temperature for 10 mins. The solids were thenremoved by filtration and then washed with Et₂O until washings ranclear. The orange solution was then concentrated in vacuo to give thedesired product 2-(di-tert-Butyl-phosphinyl)-ferrocene methanol (J) asan orange powder (2.03 g, 99%) without the need for furtherpurification. ¹H NMR (300 MHz, C₆D₆) δ_(H) 6.84 (brs, 1H), 4.69 (dd,J=13.0, 3.4 Hz, 1H), 4.49 (dd, J=13.0, 8.4 Hz, 1H), 4.23 (s, 5H),4.19-4.13 (m, 1H), 4.04 (dd, J=4.3, 2.3 Hz, 1H), 3.77 (brs, 1H), 1.45(d, J=13.8 Hz, 10H), 0.99 (d, J=13.8 Hz, 9H); ³¹P{¹H} NMR (122 MHz,C₆D₆) δ_(P) 62.27; ¹³C NMR (75 MHz, C₆D₆) δ_(C) 98.3, 72.7, 72.6, 72.5,72.3, 71.3, 70.2, 70.1, 60.7, 38.2, 37.3, 36.9, 36.1, 27.2, 26.7; HRMS(ESI μTOF) calculated for C₁₉30₉FeO₂P m/z 377.2349 found 377.2301(m/z+H).

3-((2-di-tert-butylphosphinyl)-ferrocenylmethoxy)propan-1-ol (7)

The 2-di-tert-butyl-phosphinyl-ferrocene methanol (J) (376 mg, 1 mmol, 1eq) was suspended in 1,3-propane-diol (5 cm³). The suspension wastreated with ytterbium (III) triflate (31 mg, 0.05 mmol, 5 mol %). Theflask was sealed and then heated at 100° C. for 15 mins. The flask wasallowed to cool to room temperature and the solution was diluted withH₂O (15 cm³) and EtOAc (30 cm³). The organic layer was separated and theaqueous layer back extracted with EtOAc (3×5 cm³). The combined organicswere then washed with H₂O (3×5 cm³) and brine (sat) (10 cm³). Thecombined organics were then dried over MgSO₄, filtered and concentratedin vacuo to give a brown oil. Purification by basic aluminachromatography eluting with EtOAc gave the desired product3-((2-di-tert-butylphospinyl)-ferrocenylmethoxy)propan-1l-ol (7) as anorange oil (157 mg, 36%). ¹H NMR (300 MHz, C₆D₆) δ_(H) 5.47 (brs, 1H),4.77 (d, J=10.7 Hz, 1H), 4.6-4.52 (m, 2H), 4.21-4.13 (m, 7H), 4.03-3.84(m, 3H), 3.81 (brs, 1H), 2.09-1.95 (m, 2H), 1.47 (d, J=13.6 Hz, 9H),1.05 (d, J=13.6 Hz, 9H); ³¹P{H} NMR (122 MHz, C₆D₆) δ_(H) 60.53. ¹³C NMR(75 MHz, C₆D₆) δ_(H) 92.2, 72.9, 72.8, 72.4, 72.2, 72.2, 71.4, 71.3,71.0, 71.0, 70.9, 69.1, 68.8, 59.4, 38.1, 37.3, 37.1, 36.3, 33.8, 27.5,27.1; HRMS (ESI μTOF) calculated for C₂₂H₃₅FeO₃PNa m/z 457.1639 found457.1626 (m/z+Na⁺); Electrochemical potential: 419 mV.

Example 8: Preparation of3-(2-tributylstannyl-ferrocenylmethoxy)propan-1-ol (8)

(rac)-4-(Methoxymethyl)-2-ferrocenyl-1,3-dioxane (K),(rac)-4-(Methoxymethyl)-2-(α-(tributylstannyl)-ferrocenyl)-1,3-dioxane(L) and 2-tributylstannyl ferrocene carboxaldehyde (M) were preparedaccording to the procedures in J. Org. Chem., 1997, 62, 6733-6745.

2-Tributylstannyl ferrocene carboxaldehyde (M) (447 mg, 0.88 mmol, 1 eq)was dissolved in EtOH:THF mixture (4:1) (5 cm³). The red solution wasthen treated with sodium borohydride (42 mg, 1.1 mmol, 1.3 eq) and thered solution was then stirred at room temperature for 1 hour. After thistime the now orange solution was treated with H₂O (10 cm³) and dilutedwith EtOAc (10 cm³). The organic layer was separated and the aqueouslayer back extracted with EtOAc (3×5 cm³). The combined organics werethen washed with brine (sat) (10 cm³), dried over MgSO₄, filtered andconcentrated in vacuo to give the desired alcohol as an orange oil. Theoil was suspended in 1,3-propanediol (3 cm³), then treated withytterbium (III) triflate (27 mg, 0.044 mmol, 5 mol %). The flask wassealed, then heated to 100° C. for 10 minutes. The flask was then cooledto room temperature and the solution was diluted with H₂O (10 cm³) andEtOAc (10 cm³). The organic layer was separated and the aqueous layerwas back extracted with EtOAc (3×5 cm³). The combined organics were thenwashed with brine (sat) (25 cm³), dried over MgSO₄, filtered andconcentrated in vacuo to give an orange oil. Purification by silicachromatography eluting with 20% EtOAc:n-Hex gave the desired product3-(2-tributylstannyl-ferrocenylmethoxy)propan-1-ol (8) as an orange oil(280 mg, 57%)

¹H NMR (300 MHz, C₆D₆) δ_(H) 4.43-4.33 (m, 2H), 4.28 (t, J=2.3 Hz, 1H),4.18-4.09 (m, 7H), 3.70 (q, J=5.6 Hz, 2H), 3.58 (ddd, J=9.1, 5.6, 5.5Hz, 1H), 3.49 (ddd, J=9.1, 5.6, 5.5 Hz, 1H), 1.81-1.73 (m, 6H),1.63-1.46 (m, 6H), 1.31-1.25 (m 8H), 1.07 (t, J=7.3 Hz, 9H); ¹³C NMR (75MHz, C₆D₆) δ_(H) 89.9, 76.1, 72.7, 71.4, 71.2, 69.4, 69.2, 61.9, 33.1,30.1, 28.3, 14.3, 11.2; ¹¹⁵Sn{¹H} NMR (112 MHz, C₆D₆) δ_(Sn) −20.71.HRMS (ESI μTOF) calculated for C₂₆H₄₄FeO₂SnNa m/z 587.1610 found587.1607 (m/z+Na⁺); Electrochemical potential: 303 mV.

Example 9: Preparation of3-(2-trimethylsilyl-ferrocenylmethoxy)propan-1-ol (9)

(rac)-4-(Methoxymethyl)-2-ferrocenyl-1,3-dioxane (K),(rac)-4-(Methoxymethyl)-2-(α-(trimethylsilyl)-ferrocenyl)-1,3-dioxane(N) and 2-trimethylsilyl ferrocene carboxaldehyde (O) were preparedaccording to the procedures in J. Org. Chem., 1997, 62, 6733-6745.

2-Trimethylsily ferrocene carboxaldehyde (O) (266 mg, 0.93 mmol, 1 eq)was dissolved in EtOH:THF mixture (4:1) (5 cm³). The red solution wasthen treated with sodium borohydride (44 mg, 1.2 mmol, 1.3 eq) and thenstirred at room temperature for 1 hour. After this time the now orangesolution was treated with H₂O (10 cm³) and diluted with EtOAc (10 cm³).The organic layer was separated and the aqueous layer back extractedwith EtOAc (3×5 cm³). The combined organics were then washed with brine(sat) (10 cm³), dried over MgSO₄, filtered and concentrated in vacuo togive the desired alcohol as an orange oil. The oil was suspended in1,3-propanediol (3 cm³), then treated with ytterbium (III) triflate (28mg, 0.046 mmol, 5 mol %). The flask was sealed then heated to 100° C.for 2 minutes. The flask was then cooled to room temperature, thesolution was diluted with H₂O (10 cm³) and EtOAc (10 cm³). The organiclayer was separated and the aqueous layer was back extracted with EtOAc(3×5 cm³). The combined organics were then washed with brine (sat) (25cm³), dried over MgSO₄, filtered and concentrated in vacuo to give anorange oil. Purification by silica chromatography eluting with 20%EtOAc:n-Hex gave the desired product as an orange oil (124 mg, 38%).

¹H NMR (300 MHz, C₆D₆) δ_(H) 4.29 (d, J=11.2 Hz, 1H), 4.15 (dd, J=2.3,1.3 Hz, 1H), 4.05 (t, J=2.3 Hz, 1H), 3.94-3.88 (m, 7H), 3.54 (q, J=6.1Hz, 2H), 3.41-3.26 (m, 2H) 1.67 (t, J=6.1 Hz, 1H), 1.62-1.51 (m, 2H),0.28 (s, 9H); ¹³C NMR (75 MHz, C₆D₆) δ_(C) 88.2, 74.7, 73.0, 71.6, 69.8,69.3, 68.4, 68.3, 61.0, 32.3, 0.0; HRMS (ESI μTOF) calculated forC₁₇H₂₆FeO₂SiNa m/z 369.0949 found 369.0954 (m/z+Na⁺); Electrochemicalpotential: 248 mV.

Example 10: Preparation of3-(2-tributylsilyl-ferrocenylmethoxy)propan-1-ol (10)

(rac)-4-(Methoxymethyl)-2-ferrocenyl-1,3-dioxane (K), was preparedaccording to the procedures in J. Org. Chem., 1997, 62, 6733-6745.

(rac)-4-(Methoxymethyl)-2-(α-(tributylsilyl)-ferrocenyl)-1,3-dioxane (P)was prepared via the procedure in J. Org. Chem., 1997, 62, 6733-6745using tribuylsilychloride as the electrophile.

2-tributylsilyl ferrocene carboxaldehyde (Q) was prepared via adaptingthe procedure in J. Org. Chem., 1997, 62, 6733-6745.

2-Tributylsilyl ferrocene carboxaldehyde (Q) (461 mg, 1.12 mmol, 1 eq)was dissolved in EtOH:THF mixture (4:1) (5 cm³). The red solution wasthen treated with sodium borohydride (55 mg, 1.46 mmol, 1.3 eq) and thered solution was then stirred at room temperature for 1 hour. After thistime the now orange solution was treated with H₂O (10 cm³) and dilutedwith EtOAc (10 cm³). The organic layer was separated and the aqueouslayer back extracted with EtOAc (3×5 cm³). The combined organics werethen washed with brine (sat) (10 cm³), dried over MgSO₄, filtered andconcentrated in vacuo to give the desired alcohol as an orange oil. Theoil was suspended in 1,3-propanediol (3 cm³), then treated withytterbium (III) triflate (35 mg, 0.056 mmol, 5 mol %). The flask wassealed then heated to 100° C. 20 minutes. The flask was then cooled toroom temperature, the solution was diluted with H₂O (10 cm³) and EtOAc(10 cm³). The organic layer was separated and the aqueous layer was backextracted with EtOAc (3×5 cm³). The combined organics were then washedwith brine (sat) (25 cm³), dried over MgSO₄, filtered and concentratedin vacuo to give an orange oil. Purification by silica chromatographyeluting with 20% EtOAc:n-Hex gave the desired product3-(2-tributylsilyl-ferrocenylmethoxy)propan-1-ol (10) as an orange oil(71 mg, 13%).

¹H NMR (300 MHz, C₆D₆) δ_(H) 4.46 (d, J=11.0 Hz, 1H), 4.34 (dd, J=2.3,1.2 Hz, 1H), 4.23 (t, J=2.3 Hz, 1H), 4.21-4.05 (m, 7H), 3.72 (q, J=5.5Hz, 2H), 3.63-3.44 (m, 2H), 1.84 (t, J=5.4 Hz, 1H), 1.80-1.70 (m, 2H),1.61-1.54 (m, 12H), 1.10-1.01 (m, 15H); ¹³C NMR (75 MHz, C₆D₆) δ_(C)88.8, 75.9, 73.7, 71.0, 70.9, 70.5, 69.5, 69.4, 61.9, 33.2, 27.7, 27.3,14.5; HRMS (ESI μTOF) calculated for C₂₆H₄₄FeO₂NaSi m/z 495.2357 found495.2381 (m/z+Na⁺); Electrochemical potential: 361 mV.

Example 11: Preparation of3-(2-trimethylstannyl-ferrocenylmethoxy)propan-1-ol (11)

(rac)-4-(Methoxymethyl)-2-ferrocenyl-1,3-dioxane (K), was preparedaccording to the procedures in J. Org. Chem., 1997, 62, 6733-6745.

(rac)-4-(Methoxymethyl)-2-(α-(trimethylstannyl)-ferrocenyl)-1,3-dioxane(R) was prepared via the procedure in J. Org. Chem., 1997, 62, 6733-6745using trimethyltinchloride as the electrophile. 2-trimethylstannylferrocene carboxaldehyde (T) was prepared via adapting the procedure inJ. Org. Chem., 1997, 62, 6733-6745.

2-Trimethylstannyl ferrocene carboxaldehyde (T) (356 mg, 1.12 mmol, 1eq) was dissolved in EtOH:THF mixture (4:1) (5 cm³). The red solutionwas then treated with sodium borohydride (45 mg, 1.2 mmol, 1.3 eq) andthe red solution was then stirred at room temperature for 1 hour. Afterthis time the now orange solution was treated with H₂O (10 cm³) anddiluted with EtOAc (10 cm³). The organic layer was separated and theaqueous layer back extracted with EtOAc (3×5 cm³). The combined organicswere then washed with brine (sat) (10 cm³), dried over MgSO₄, filteredand concentrated in vacuo to give the desired alcohol as an orange oil.The oil was suspended in 1,3-propanediol (3 cm³), then treated withytterbium (III) triflate (29 mg, 0.048 mmol, 5 mol %). The flask wassealed then heated to 100° C. 20 minutes. The flask was then cooled toroom temperature, the solution was diluted with H₂O (10 cm³) and EtOAc(10 cm³). The organic layer was separated and the aqueous layer was backextracted with EtOAc (3×5 cm³). The combined organics were then washedwith brine (sat) (25 cm³), dried over MgSO₄, filtered and concentratedin vacuo to give an orange oil. Purification by silica chromatographyeluting with 20% EtOAc:n-Hex gave the desired product3-(2-trimethylstannyl-ferrocenylmethoxy)propan-1-ol (11) as an orangeoil (190 mg, 48%)

¹H NMR (300 MHz, C₆D₆) δ_(H) 4.16-4.10 (m, 2H), 4.05 (t, J=2.3 Hz, 1H),3.92-3.86 (m, 6H), 3.85 (dd, J=2.2, 1.1 Hz, 1H), 3.45 (dd, J=11.3, 5.6Hz, 2H), 3.27 (ddt J=20.7, 9.1, 5.6 Hz, 2H), 1.54-1.45 (2H, m), 0.21(ss, 9H); ¹³C NMR (75 MHz, C₆D₆) δ_(C) 90.1, 75.8, 72.7, 71.1, 70.9,70.7, 69.1, 61.7, 33.1, −7.8; ¹¹⁵Sn{¹H} NMR (112 MHz, C₆D₆) δ_(Sn)−9.03. HRMS (ESI μTOF) calculated for C₁₇H₂₆FeO₂NaSn m/z 461.0201 found461.0221 (m/z+Na⁺); Electrochemical potential: 207 mV.

Example 12: Preparation of 3-(2-vinyl-ferrocenylmethoxy)propan-1-ol (12)

(rac)-4-(Methoxymethyl)-2-ferrocenyl-1,3-dioxane (K) and(rac)-4-(Methoxymethyl)-2-(α-formyl-ferrocenyl)-1,3-dioxane (U) wereprepared according to the procedures in J. Org. Chem., 1997, 62,6733-6745.

(rac)-4-(Methoxymethyl)-2-(α-vinyl-ferrocenyl)-1,3-dioxane (V)

(rac)-4-(Methoxymethyl)-2-(α-formyl-ferrocenyl)-1,3-dioxane (U) (481 mg,1.4 mmol, 1 eq) was dissolved in dry THF (15 cm³) and then treated withmethyltriphenyl phosphonium bromide (999 mg, 2.8 mmol, 2 eq) andpotassium tert-butoxide (313 mg, 2.8 mmol, 2 eq). The mixture was thenstirred at room temperature for 3 hours. After this time the reactionwas quenched by addition of H₂O (10 cm³). The organic layer was thenseparated and the aqueous layer back extracted with EtOAc (3×5 cm³). Thecombined organics were then dried over MgSO₄, filtered and concentratedin vacuo to give an orange oil. Purification by silica chromatographyeluting with 20% EtOAc:n-Hex to give the desired product(rac)-4-(Methoxymethyl)-2-(α-vinyl-ferrocenyl)-1,3-dioxane (V) as anorange oil (137 mg, 29%).

¹H NMR (300 MHz, C₆D₆) δ_(H) 6.80 (dd, J=17.6, 10.9 Hz, 1H), 5.42 (dd,J=17.6, 1.8 Hz, 1H), 5.35 (s, 1H), 5.08 (dd, J=10.9, 1.8 Hz, 1H), 4.57(dd, J=2.4, 1.5 Hz, 1H), 4.25 (dd, J=2.4, 1.5 Hz, 1H), 4.06 (s, 5H),3.94 (t, J=2.4 Hz, 1H), 3.88 (ddd, J=11.5, 5.5, 1.2 Hz, 1H), 3.42 (ddd,J=10.1, 11.5, 2.6 Hz, 1H), 3.25 (dd, J=10.1, 5.5 Hz, 1H), 3.09-3.03 (m,4H), 1.65-1.46 (m, 1H), 1.03-0.92 (m, 1H); ¹³C NMR (75 MHz, C₆D₆) δ_(H)134.7, 112.4, 100.5, 85.4, 82.8, 76.7, 76.2, 70.8, 69.1, 68.1, 67.1,66.90, 59.4, 28.8; HRMS (ESI TOF) calculated for C₁₈H₁₂₂FeO₃Na m/z365.0816 found 365.0818 (m/z+Na⁺).

3-(2-Vinyl-ferrocenylmethoxy)propan-1-ol (12)

The (rac)-4-(methoxymethyl)-2-(α-vinyl-ferrocenyl)-1,3-dioxane (V) (137mg, 0.4 mmol, 1 eq) was placed in a Schlenk tube withpara-toluenesulfonic acid monohydrate (200 mg, 1 mmol, 2.5 eq). Theflask was sealed then evacuated and back filled with argon four times.The flask was then charged with CH₂Cl₂ (7 cm³) and H₂O (3 cm³). Thebi-phasic mixture was then stirred vigorously for 18 hours. After thistime the organic layer was separated and the aqueous layer backextracted with CH₂Cl₂ (3×5 cm³). The combined organics were then washedwith H₂O (10 cm³), then dried over MgSO₄, filtered and concentrated invacuo to give the aldehyde as a red oil. This was then taken up inEtOH:THF (4:1) (5 cm³) and treated with sodium borohydride (27 mg, 0.48mmol, 1.2 eq). The red solution was then stirred at room temperature for30 minutes. At this point the orange solution was treated with NaHCO₃(sat) (10 cm³) and then diluted with EtOAc (10 cm³). The organic layerwas separated and the aqueous layer back extracted with EtOAc (3×5 cm³).The combined organics were then washed with H₂O (10 cm³), dried overMgSO₄, filtered and then concentrated in vacuo to give the targetalcohol as an orange oil. This was then dissolved in 1,3-propandiol (3cm³), the solution was treated with ytterbium (III) triflate (12 mg,0.02 mmol, 5 mol %). The flask was sealed and then heated at 100° C. for15 minutes. The flask was then allowed to cool to room temperature, thebrown solution was then diluted with EtOAc (10 cm³) and H₂O (10 cm³).The organic layer was separated and the aqueous layer back extractedwith EtOAc (3×5 cm³). The combined organics were washed with H₂O (25cm³), dried over MgSO₄, filtered and concentrated in vacuo to give anorange oil. Purification by silica chromatography eluting with 40%EtOAc:n-Hex to give the desired product3-(2-Vinyl-ferrocenylmethoxy)propan-1-ol (12) as an orange oil (3 mg,2.5%).

¹H NMR (300 MHz, C₆D₆) δ_(H) 6.47 (dd, J=17.5, 10.9 Hz, 1H), 5.39 (dd,J=17.5, 1.7 Hz, 1H), 5.05 (dd, J=10.9, 1.7 Hz, 1H), 4.42-4.29 (m, 2H),4.08-3.91 (m, 6H), 3.87 (s, 5H), 3.54 (dd, J=11.2, 5.5 Hz, 2H),3.41-3.28 (m, 2H), 1.70 (t, J=5.4 Hz, 1H), 1.53 (dt, J=7.6, 5.8 Hz, 2H);¹³C NMR (75 MHz, C₆D₆) δ_(C) 133.7, 112.7, 82.9, 82.3, 71.5, 70.3, 69.1,68.4, 68.2, 67.1, 64.7, 61.9, 33.1; HRMS (ESI TOF) calculated forC₁₆H₂₀FeO₂Na m/z 323.0630 found 323.0646 (m/z+Na⁺); Electrochemicalpotential: 220 mV.

Example 13: Preparation of 3-(2-iodo-ferrocenylmethoxy)propan-1-ol (13)

3-(2-tributylsilyl-ferrocenylmethoxy)propan-1-ol (12) (265 mg, 0.47mmol, 1 eq) was dissolved in CH₂Cl₂ (2.5 cm³) and then treated withiodine (130 mg, 0.51 mmol, 1.1 eq). The dark brown solution was thenstirred at room temperature for 16 hours. After this time the reactionwas quenched by addition of sodium thiosulphate (sat) (5 cm³). Theorganic layer was separated and the aqueous layer back extracted withCH₂Cl₂ (3×5 cm³). The combined organics were then dried over MgSO₄,filtered and concentrated in vacuo to give an orange oil. Purificationby silica chromatography eluting with 30% EtOAc:n-Hex to give thedesired product 3-(2-iodo-ferrocenylmethoxy)propan-1-ol (13) as anorange oil (15 mg, 8%).

¹H NMR (300 MHz, C₆D₆) δ_(H) 4.36-4.28 (m, 2H), 4.19 (d, J=11.5 Hz, 1H),4.14 (dd, J=2.5, 1.3 Hz, 1H), 4.01 (s, 5H), 3.91 (t, J=2.5 Hz, 1H), 3.71(dd, J=10.6, 5.3 Hz, 2H), 3.61-3.47 (m, 2H), 1.76-1.65 (m, 2H); ¹³C NMR(75 MHz, C₆D₆) 5c 85.8, 75.6, 72.1, 69.7, 69.7, 69.6, 68.9, 61.9, 45.3,33.1, 32.3, 23.4, 14.7; HRMS (ESI TOF) calculated for C₁₄H₁₇FeO₂INa m/z422.9520 found 422.9538 (m/z+Na⁺); Electrochemical potential: 355 mV.

Example 14: Preparation of 2-(3-ferrocenylpropoxy)ethanol (14)

Ethyl-3-ferrocenyl acrylate (W) was prepared according to the procedurein Tetrahedron, 2009, 65, 672-676.

Ethyl-3-ferrocenyl Propanoate (X)

The ethyl-3-ferrocenyl acrylate (W) (7.43 g, 23.3 mmol, 1 eq) wasdissolved in MeOH (125 cm³) and cooled to 0° C. Once cold the palladiumon carbon (10% wt) (1.5 g) and ammonium formate (5.87 g, 93.2 mmol, 4eq) were added sequentially. The black suspension was allowed to warm toroom temperature and then stirred for 1 hour. The suspension wasfiltered through celite, and the solids washed with MeOH (100 cm³). Theorange solution was then concentrated in vacuo to give an orange solid,which was then taken up in EtOAc (100 cm³) and NaHCO₃ (50 cm³). Theorganic layer was separated and the aqueous layer back extracted withEtOAc (3×50 cm³). The combined organics were then dried over MgSO₄,filtered and concentrated in vacuo to give the desired productethyl-3-ferrocenyl propanoate (X) as an orange oil (4.86 g, 73%) withoutthe need for further purification.

¹H NMR (250 MHz, CDCl₃); δ_(H): 4.2 (d, J=7.0, 2H), 4.09 (s, 2H), 4.02(s, 7H), 2.93-2.87 (m, 2H), 2.54-2.49 (m, 2H), 2.52 (1H, br s), 1.37 (t,J=7.0 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃); δ_(C): 170.5, 77.5, 77.0, 76.6,69.8, 61.3, 34.8, 23.2, 15.3.

Ferrocene Propanol (Y)

To a suspension of lithium aluminium hydride (1.94 g, 51 mmol, 3 eq) indry Et₂O (120 cm³) at 0° C. was added ethyl-3-ferrocenyl propanoate (X)(4.86 g, 17 mmol, 1 eq) in dry Et₂O (30 cm³) dropwise over a 25 minuteperiod. Once the addition was complete the suspension was allowed towarm to room temperature and stirred for 1 hour. After this time theflask was cooled to 0° C. and the reaction was quenched by sequentialdropwise addition of H₂O (1.9 cm³), 15% NaOH (aq) (1.9 cm³) and H₂O (5.7cm³). The yellow suspension was then allowed to warm to room temperatureand was stirred for 10 minutes. The suspension was filtered, and solidswashed with Et₂O (75 cm³) until the washing ran clear. The orangesolution was dried over MgSO₄, filtered and concentrated in vacuo togive ferrocene propanol (Y) as an orange oil (4.19 g, 99%) without theneed for further purification.

¹H NMR (300 MHz, CDCl₃); δ_(H): 4.19 (s, 9H), 3.65 (t, J=6.0 Hz, 2H),2.55 (d, J=6.0 Hz, 2H), 1.70 (t, J=6.0 Hz, 2H); ¹³C NMR (75 MHz, CDCl₃);δ_(C): 77.8, 77.6, 77.4, 76.9, 63.4, 38.5, 29.4.

Ethyl 2-ferrocenethoxyacetate (Z)

The ferrocene propanol (Y) (1.93 g, 7.9 mmol, 1 eq) was placed in around bottomed flask and treated with ethyl diazoacetate (552 μl, 5.26mmol, 0.66 eq) and indium (III) chloride (464 mg, 2.1 mmol, 40 mol %).The slurry was allowed to stir at room temperature under nitrogen for 16hours. After this time the slurry was diluted with EtOAc (25 cm³) andH₂O (25 cm³). The organic layer was separated and the aqueous layer backextracted with EtOAc (3×20 cm³). The combined organics were washed withbrine (sat) (50 cm³), dried over MgSO₄, filtered and concentrated invacuo to give an orange oil. Purification by silica chromatographyeluting with 15% EtOAc:n-Hex gave the desired product ethyl2-ferrocenethoxyacetate (Z) as an orange oil (993 mg, 57%).

¹H NMR (300 MHz, C₆D₆) δ_(H) 4.11-4.06 (m, 8H), 4.04-3.98 (m, 5H), 3.94(s, 2H), 2.50 (dd, J=8.7, 6.8 Hz, 2H), 1.94-1.80 (m, 2H), 1.00 (t, J=7.1Hz, 3H); ¹³C NMR (75 MHz, C₆D₆) δ_(H) 170.5, 89.2, 71.6, 69.2, 68.9,68.3, 67.9, 60.6, 32.0, 26.6, 14.5.

2-(3-Ferrocenylpropoxy)ethanol (14)

To a suspension of lithium aluminium hydride (343 mg, 9 mmol, 3 eq) indry Et₂O (10 cm³) at 0° C. was added the ethyl 2-ferrocenethoxyacetate(Z) (993 mg, 3 mmol, 1 eq). in dry Et₂O (5 cm³) dropwise over a 5 minuteperiod. The suspension was allowed to warm to room temperature andstirred for 30 minutes. After this time the flask was cooled to 0° C.and the reaction was quenched by sequential dropwise addition of H₂O(343 μl), 15% NaOH (aq) (343 μl) and H₂O (1.2 cm³). The yellowsuspension was then allowed to warm to room temperature and was stirredfor 10 minutes. The suspension was filtered, and solids washed with Et₂O(25 cm³) until the washings ran clear. The orange solution was driedover MgSO₄, filtered and concentrated in vacuo to give an orange oil.Purification by silica chromatography eluting with 20% EtOAc:n-Hex gavethe desired product 2-(3-ferrocenylpropoxy)ethanol as an orange oil (739mg, 85%).

¹H NMR (300 MHz, C₆D₆) δ_(H) 3.90 (s, 5H), 3.85 (s, 4H), 3.47-3.35 (m,2H), 3.11 (dd, J=10.4, 5.4 Hz, 4H), 2.29-2.14 (m, 2H), 1.67-1.47 (m,3H); ¹³C NMR (75 MHz, C₆D₆) δ_(C) 89.1, 72.6, 71.1, 69.2, 68.8, 67.9,62.3, 31.9, 26.8; HRMS (ESI μTOF) calculated for C₁₅H₂₁FeO₂ m/z 289.1553found 289.0987 (m/z+H); Electrochemical potential: 114 mV.

Example 15: Preparation of2-(3-(2-tert-butylthio)-ferrocenylpropoxy)ethanol (15)

2-tert-butylthio-ferrocene Carboxaldehyde (AA)

The 2-tert-butylthio ferrocene methanol (G) (741 mg, 2.4 mmol, 1 eq) wasplaced in a Schlenk tube with barium manganate (2.49 g, 9.7 mmol, 4 eq).The flask was sealed, then evacuated and backfilled under argon fourtimes. The flask was then charged with benzene (15 cm³). The resultingdark blue slurry was then stirred at room temperature for 16 hours.After this time the slurry was filtered through celite and solids washedwith Et₂O (25 cm³) until washings ran clear. The resulting red solutionwas concentrated in vacuo to give the desired aldehyde2-tert-butylthio-ferrocene carboxaldehyde (AA) as a red oil (668 mg,92%) without the need for further purification.

¹H NMR (300 MHz, C₆D₆) δ_(H) 10.66 (s, 1H), 5.09-5.01 (m, 1H), 4.45 (dd,J=2.4, 1.7 Hz, 1H), 4.19 (dd, J=2.4, 1.7 Hz, 1H), 4.04 (s, 5H), 1.14 (s,9H); ¹³C NMR (75 MHz, C₆D₆) δ_(C) 193.4, 82.3, 81.7, 81.1, 73.2, 71.5,69.6, 45.9, 30.9; HRMS (ESI μTOF) calculated for C₁₅H₁₈FeOSNa m/z325.0325 found 325.0325 (m/z+Na⁺).

Ethyl-3-(2-tert-butylthio-ferrocenyl) Acrylate (AB)

To a suspension of sodium hydride (60% dispersion in oil) (109 mg, 2.85mmol, 1.3 eq) in dry THF (10 cm³) at 0° C. was addedtriethylphosphonacetate (571 ml, 2.85 mmol, 1.3 eq) dropwise over a 5minute period. Once addition was complete the solution was allowed towarm to room temperature and stirred for 30 mins. After this time thesolution was cooled to 0° C. Once cold the 2-tert-butyl-ferrocenecarboxaldehyde (AA) (668 mg, 2.2 mmol, 1 eq) in dry THF (5 cm³) wasadded dropwise over a 5 min period. Once addition was complete the flaskwas allowed to warm to room temperature and was stirred for anadditional 30 minutes. After this time the reaction was quenched byaddition of H₂O (20 cm³). The organic layer was then separated and theaqueous layer back extracted with EtOAc (3×5 cm³). The combined organicswere then washed with brine (sat) (10 cm³), dried over MgSO₄, filteredand concentrated in vacuo to give a red oil. Purification by silicachromatography eluting with 5% EtOAc:n-Hex gave the desired productethyl-3-(2-tert-butylthio-ferrocenyl) acrylate (AB) as a red oil (823mg, 99%).

¹H NMR (300 MHz, C₆D₆) δ_(H) 8.50 (d, J=16.0 Hz, 1H), 6.48 (d, J=16.0Hz, 1H), 4.50 (dd, J=2.5, 1.4 Hz, 1H), 4.44 (dd, J=2.5, 1.4 Hz, 1H),4.23 (qd, J=7.1, 2.2 Hz, 2H), 4.16 (t, J=2.5 Hz, 1H), 4.01 (s, 4H), 1.21(s, 9H), 1.14 (t, J=7.1 Hz, 3H); ¹³C NMR (75 MHz, C₆D₆) δ_(H) 185.5,167.2, 144.8, 116.4, 83.4, 79.9, 72.6, 71.7, 71.4, 67.3, 60.5, 51.1,46.2, 40.4, 31.1, 20.3, 14.8, 10.0; HRMS (ESI μTOF) calculated forC₁₉H₂₄FeO₂SNa m/z 395.0744 found 395.0748 (m/z+Na⁺).

Ethyl-3-(2-tert-butylthioferrocenyl) Propanoate (AC)

The ethyl-3-(2-tert-butylthio-ferrocenyl) acrylate (AB) (823 mg, 2.2mmol, 1 eq) was dissolved in methanol (15 cm³) and cooled to 0° C. Oncecold, palladium on carbon (10% wt) (1 g) and ammonium formate (831 mg,13.2 mmol, 6 eq) were added. The suspension was allowed to warm to roomtemperature and stirred for 4 hours. After this time suspension wasfiltered through celite and the solids were washed with methanol (25cm³) until washings ran clear. The orange solution was then concentratedin vacuo to give an orange solid. This was partitioned between EtOAc (25cm³) and NaHCO₃ (sat) (25 cm³). The organic layer was separated and theaqueous layer back extracted with EtOAc (3×5 cm³). The combined organicswere dried over MgSO₄, filtered and concentrated in vacuo to give thedesired material Ethyl-3-(2-tert-butylthioferrocenyl) propanoate (AC) asan orange oil (617 mg, 75%) without the need for further purification.

¹H NMR (300 MHz, C₆D₆) δ_(H) 4.42 (dd, J=2.3, 1.4 Hz, 1H), 4.14-4.10 (m,3H), 4.08 (s, 5H), 4.04 (t, J=2.5 Hz, 1H), 3.17 (ddd, J=15.4, 8.9, 6.7Hz, 1H), 3.00 (ddd, J=15.4, 8.9, 6.7 Hz, 1H), 2.79-2.60 (m, 2H), 1.26(s, 9H), 1.09 (t, J=7.1 Hz, 3H); ¹³C NMR (75 MHz, C₆D₆) δ_(C) 173.1,92.3, 77.3, 76.9, 70.7, 69.0, 68.4, 60.6, 45.9, 35.6, 31.4, 23.8, 14.7;HRMS (ESI μTOF) calculated for C₁₉H₂₆FeO₂SNa m/z 397.0900 found 397.0917(m/z+Na⁺).

2-tert-butylthioferrocene Propanol (AD)

To a suspension of lithium aluminium hydride (188 mg, 4.9 mmol, 3 eq) indry Et₂O (3.5 cm³) at 0° C. was addedethyl-3-(2-tert-butylthioferrocenyl) propanoate (AC) (617 mg, 16 mmol, 1eq) in dry Et₂O (4 cm³) was added dropwise over a 2 minute period. Oncethe addition was complete the suspension was allowed to warm to roomtemperature and stirred for 30 mins. After this time the flask wascooled to 0° C. and the reaction was quenched by sequential dropwiseaddition of H₂O (188 μl), 15% NaOH (aq) (188 μl) and H₂O (546 μl). Theyellow suspension was then allowed to warm to room temperature and wasstirred for 10 minutes. The suspension was filtered, and solids washedwith Et₂O (15 cm³) until the washing ran clear. The orange solution wasdried over MgSO₄, filtered and concentrated in vacuo to give2-tert-butylthioferrocene propanol (AD) as an orange oil (381 mg, 66%)without the need for further purification.

¹H NMR (300 MHz, C₆D₆) δ_(H) 4.45 (dd, J=2.4, 1.4 Hz, 1H), 4.17-4.15 (m,1H), 4.14 (s, 5H), 4.09 (t, J=2.5 Hz, 1H), 3.56 (t, J=6.4 Hz, 2H), 2.79(ddd, J=14.9, 11.2, 5.6 Hz, 1H), 2.58 (ddd, J=14.9, 11.2, 5.6 Hz, 1H),1.97-1.71 (m, 2H), 1.30 (s, 9H); ¹³C NMR (75 MHz, C₆D₆) δ_(C) 93.5,77.2, 76.7, 70.7, 69.1, 68.3, 63.2, 45.8, 34.0, 31.8, 24.8; HRMS (ESIμTOF) calculated for C₁₇H₂₄FeOSNa m/z 355.0794 found 355.0780 (m/z+Na⁺).

Ethyl 2-(2-tert-butylthioferrocene)ethoxyacetate (AE)

The 2-tert-butylthioferrocene propanol (AD) (483 mg, 1.29 mmol, 1 eq)was placed in a round bottomed flask and treated with ethyl diazoacetate(552 μl, 5.26 mmol, 4 eq) and indium (III) chloride (114 mg, 0.52 mmol,40 mol %). The slurry was allowed to stir at room temperature undernitrogen for 16 hours. After this time the slurry was diluted with EtOAc(25 cm³) and H₂O (15 cm³).

The organic layer was separated and the aqueous layer back extractedwith EtOAc (3×5 cm³). The combined organics were washed with brine (sat)(10 cm³), dried over MgSO₄, filtered and concentrated in vacuo to givean orange oil. Purification by silica chromatography eluting with 10%EtOAc:n-Hex gave the desired product as an orange oil (164 mg, 30%).

¹H NMR (300 MHz, C₆D₆) δ_(H) 4.45 (dd, J=2.5, 1.4 Hz, 1H), 4.19 (dd,J=2.5, 1.4 Hz, 1H), 4.14 (s, 5H), 4.09-3.97 (m, 9H), 3.59 (td, J=8.6,2.3 Hz, 2H), 2.90 (ddd, J=15.0, 11.1, 5.6 Hz 1H), 2.69 (ddd, J=15.0,11.1, 5.6 Hz 1H), 2.18-1.91 (m, 2H), 1.30 (s, 9H), 1.02 (t, J=6.3 Hz,3H); ¹³C NMR (75 MHz, C₆D₆) δ_(C) 170.6, 93.5, 77.2, 76.7, 72.2, 70.7,69.2, 68.9, 68.3, 61.0, 60.7, 45.8, 31.5, 31.2, 25.0, 14.7, 14.6; HRMS(ESI TOF) calculated for C₂₁H₃₀FeO₃SNa m/z 441.1162 found 441.1179(m/z+Na⁺).

2-(3-(2-tert-butylthio)-ferrocenylpropoxy)ethanol (15)

To a suspension of lithium aluminium hydride (44 mg, 1.17 mmol, 3 eq) indry Et₂O (2 cm³) at 0° C. was added the ethyl2-(2-tert-butylthioferrocene)ethoxyacetate (AE) (164 mg, 0.39 mmol, 1eq). in dry Et₂O (1 cm³) dropwise over a 2 minute period. The suspensionwas allowed to warm to room temperature and stirred for 30 minutes.After this time the flask was cooled to 0° C. and the reaction wasquenched by sequential dropwise addition of H₂O (44 μl), 15% NaOH (aq)(44 μl) and H₂O (132 cm³). The yellow suspension was then allowed towarm to room temperature and was stirred for 10 minutes. The suspensionwas filtered, and solids washed with Et₂O (10 cm³) until the washingsran clear. The orange solution was dried over MgSO₄, filtered andconcentrated in vacuo to give an orange oil. Purification by silicachromatography eluting with 20% EtOAc:n-Hex gave the desired product2-(3-(2-tert-butylthio)-ferrocenylpropoxy)ethanol (15) as an orange oil(41 mg, 28%).

¹H NMR (300 MHz, C₆D₆) δ_(H) 4.46 (dd, J=2.6, 1.4 Hz, 1H), 4.19 (dd,J=2.6, 1.4 Hz, 1H), 4.15 (s, 5H), 4.10 (t, J=2.6 Hz, 1H), 3.67 (dd,J=9.7, 5.3 Hz, 2H), 3.43 (t, J=6.5 Hz, 2H), 3.37 (t, J=6.5 Hz 2H), 2.81(ddd, J=14.9, 11.1, 5.7 Hz, 1H), 2.63 (ddd, J=14.9, 11.1, 5.7 Hz, 1H),2.13 (t, J=5.9 Hz, 1H), 2.09-1.85 (m, 2H), 1.30 (s, 9H); ¹³C NMR (75MHz, C₆D₆) δ_(C) 93.4, 77.2, 76.8, 72.8, 71.8, 70.7, 69.1, 68.3, 62.3,45.8, 31.5, 31.2, 25.1; HRMS (ESI TOF) calculated for C₁₉H₂₈FeO₂SNa m/z399.1057 found 399.1063 (m/z+Na⁺); Electrochemical potential: 297 mV.

Example 16: Preparation of2-(3-(2-tert-butylsulfinyl)-ferrocenylpropoxy)ethanol (16)

The 2-(3-(2-tert-butylthio)-ferrocenylpropoxy)ethanol (15) (40 mg, 0.11mmol, 1 eq) was dissolved in CH₂Cl₂ (2 cm³), the flask was then placedunder a nitrogen atmosphere and cooled to 0° C. Once cold3-chloro-perbenzoic acid (22 mg, 0.127 mmol, 1.2 eq) was added in oneportion. The solution was then stirred at 0° C. for 15 minutes. Afterthis time TLC analysis indicated full consumption of the startingmaterial. The reaction was then quenched by addition of NaHCO₃ (sat) (5cm³) and stirred vigorously for 5 minutes. After this time the organiclayer was separated and aqueous layer extracted with CH₂Cl₂ (3×5 cm³).The combined organic were then washed with brine (sat) (10 cm³), driedover MgSO₄, filtered and concentrated in vacuo to give a dark brown oil.Purification by silica chromatography eluting with EtOAc gave thedesired product 2-(3-(2-tert-butylsulfinyl)-ferrocenylpropoxy)ethanol(16) as a yellow solid (12 mg, 29%).

¹H NMR (300 MHz, C₆D₆) δ_(H) 4.81 (s, 1H), 4.41 (s, 5H), 4.10 (t, J=2.4Hz, 1H), 4.07 (s, 1H), 3.75 (t, J=6.2 Hz, 2H), 3.46 (t, J=6.2 Hz, 2H),3.40 (t, J=6.2 Hz, 2H), 2.68 (ddd, J=15.2, 11.5, 4.7 Hz 1H), 2.34 (ddd,J=15.2, 11.5, 4.7 Hz 1H), 2.0-1.91 (m, 1H), 1.90-1.78 (m, 1H), 1.13 (s,9H); ¹³C NMR (75 MHz, C₆D₆) δ_(C) 92.1, 88.0, 73.0, 71.4, 69.6, 68.6,65.9, 62.3, 56.1, 30.6, 25.5, 23.4; HRMS (ESI μTOF) calculated forC₁₉H₂₈FeO₃SNa m/z 415.1006 found 415.1010 (m/z+Na⁺); Electrochemicalpotential: 397 mV.

Example 17: Preparation of2-(3-(2-tert-butylsulfonyl)-ferrocenylpropoxy)ethanol (17)

2-(3-(2-tert-butylsulfinyl)-ferrocenylpropoxy)ethanol (16) (12 mg, 0.03mmol, 1 eq) was dissolved in CH₂Cl₂ (1 cm³), placed under a nitrogenatmosphere and cooled to 0° C. Once cold, 3-chloro-perbenzoic acid (6.3mg, 0.036 mmol, 1.2 eq) was added in one portion. The solution was thenstirred at 0° C. for 15 minutes. After this time TLC analysis indicatedfull consumption of the starting material. The reaction was thenquenched by addition of NaHCO₃ (sat) (5 cm³) and stirred vigorously for5 minutes. After this time the organic layer was separated and aqueouslayer extracted with CH₂Cl₂ (3×5 cm³). The combined organics were thenwashed with brine (sat) (10 cm³), dried over MgSO₄, filtered andconcentrated in vacuo to give a dark brown oil. Purification by silicachromatography eluting with EtOAc gave the desired product2-(3-(2-tert-butylsulfonyl)-ferrocenylpropoxy)ethanol (17) as a yellowoil (4 mg, 33%).

¹H NMR (300 MHz, C₆D₆) δ_(H) 4.58 (dd, J=2.4, 1.6 Hz, 1H), 4.41 (s, 4H),4.09 (dd, J=2.4, 1.6 Hz, 1H), 4.01 (t, J=2.4 Hz, 1H), 3.65 (t, J=4.5 Hz,2H), 3.4-3.34 (m, 4H), 3.15 (ddd, J=15.3, 11.9, 5.2 1H), 2.65 (ddd,J=15.3, 11.9, 5.2, Hz 1H), 2.0-1.73 (m, 4H), 1.23 (s, 9H); ¹³C NMR (75MHz, C₆D₆) δ_(H) 101.9, 91.8, 88.7, 82.9, 75.7, 72.8, 72.7, 72.0, 71.5,71.0, 69.3, 62, 59.4, 36.2, 30.9, 24.7, 24.1, 24.0; HRMS (ESI TOF)calculated for C₁₉H₂₈FeO₄SNa m/z 431.0955 found 431.0954 (m/z+Na⁺);Electrochemical potential: 489 mV.

Example 18: Preparation of2-cyanoethyl-(2-(3-ferrocenylpropoxy)ethanol)diisopropyl-phosphoramidite

To an oven dried 100 cm³ round bottomed flask equipped with a magneticstirrer was added the 2-(3-ferrocenylpropoxy)ethanol (14) (753 mg, 2.6mmol, 1 eq). The flask was then sealed and purged with N₂. The yellowpowder was then dissolved in dry THF (25 cm³) to give an orangesolution, this was then immediately treated with DIPEA (1.95 ml, 11.2mmol, 4.3 eq). The 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (1g, 4.2 mmol, 1.6 eq) was then added to the 14 solution over a 2 minperiod. Once complete the orange solution was allowed to stir for 10mins. H₂O (200 μl) was then added and the orange solution stirred for afurther 30 mins under nitrogen. The reaction was then quenched byaddition of EtOAc:TEA (1:1, 25 cm³). The mixture was then washed withNaHCO₃ (sat) (10 cm³) and brine (sat) (10 cm³). The orange organic layerwas then dried over Na₂SO₄, filtered, then concentrated in vacuo to givea yellow oil. Purification by silica chromatography (ø5×10 cm³, CH₂Cl₂wet load) eluting with 10% EtOAc:n-Hex+1% triethylamine under a nitrogenexit stream gave the desired product2-cyanoethyl-(2-(3-ferrocenylpropoxy)ethanol)diisopropylphosphoramiditeas an orange oil (946 mg, 78%).

¹H NMR (300 MHz, CDCl₃) δ_(H) 4.09 (s, 5H), 4.06-4.04 (m, 4H), 3.88-3.79(m, 2H), 3.60 (t, J=5.4 Hz, 2H), 3.48 (t, J=6.5 Hz, 2H), 2.65 (t, J=6.6Hz, 2H), 2.44-2.32 (m, 2H), 1.79 (dt, J=14.1, 6.5 Hz, 2H), 1.19 (d,J=6.8 Hz, 12H); ¹³C NMR (75 MHz, CDCl₃) δ_(C) 117.7, 88.7, 70.9, 70.8,70.7, 68.4, 68.0, 67.1, 62.8, 62.5, 58.6, 58.3, 43.1, 42.9, 31.0, 26.0,24.7, 24.6, 24.6, 24.5, 20.4, 20.3; ³¹P{¹H} NMR (122 MHz, CDCl₃) δp149.18. HRMS (ESI μTOF) calculated for C₂₄H₃₇FeN₂O₃PNa m/z 511.1886found 511.1893 (m/z+Na⁺).

Example 19:2-cyanoethyl-(3-(Nonamethylferrocenylmethoxy)propan-1-ol)di-iso-propyl-phosphoramidite

To an oven dried 100 cm³ round bottomed flask equipped with a magneticstirrer was added the 3-(nonamethylferrocenylmethoxy)propan-1-ol (2)(1.12 g, 2.8 mmol, 1 eq). The flask was then sealed and purged with N₂.The yellow powder was then dissolved in dry THF (25 cm³) to give anorange solution, this was then immediately treated with DIPEA (1.95 ml,11.2 mmol, 4 eq). The 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite(1 g, 4.2 mmol, 1.5 eq) was then added to the 2 solution over a 2 minperiod. Once complete the orange solution was allowed to stir for 10mins. H₂O (200 μl) was then added and the orange solution stirred for afurther 30 mins under nitrogen. The reaction was then quenched byaddition of EtOAc:TEA (1:1, 25 cm³). The mixture was then washed withNaHCO₃ (sat) (10 cm³) and brine (sat) (10 cm³). The orange organic layerwas then dried over Na₂SO₄, filtered, then concentrated in vacuo to givea yellow oil. Purification by silica chromatography (05×10 cm³, CH₂Cl₂wet load) eluting with 10% EtOAc:n-Hex+1% triethylamine under a nitrogenexit stream gave the desired product as an orange oil (995 mg, 59%).

¹H NMR (300 MHz, C₆D₆) δ_(H) 4.43 (d, 1H, J=11.3), 4.38 (d, 1H, J=11.3)3.99-3.75 (m, 2H), 3.72-3.56 (m, 4H), 3.52-3.33 (m, 2H), 2.0-1.77 (m,10H), 1.73 (s, 21H), 1.23 (t, J=6.4, 6H); ³¹P {¹H}NMR (122 MHz, C₆D₆)δ_(P) 148.75 (s); ¹³C NMR (75 MHz, C₆D₆) δ_(C) 90.4, 80.4, 80.2, 79.2,78.0, 71.9, 66.7, 66.5, 63.9, 61.3, 59.1, 47.2, 43.7, 43.6, 37.3, 32.7,30.8, 25.1, 25.0, 25.0, 20.4, 16.6, 10.2, 10.1, 9.93; HRMS (ESI μTOF)calculated for C₃₂H₅₃FeNO₃PNa m/z 623.3041 found 623.3031 (m/z+Na⁺).

The electrochemical data show that compounds of the invention provideuseful electrochemically active labels. The labels may be used toprovide an electrochemical signal within a desired range of values. Theymay be useful as alternative labels to other labelling compounds withsimilar potential values, for example, where those other labellingcompounds have disadvantageous properties in the assay in question, forexample, incompatibility with impurities or other components present inthe assay or incompatibility with the measurement conditions, any ofwhich could affect measurement sensitivity. As well, or instead, theymay be used with one or more other labels in a multiplex assay in whichmore than one label is present to provide two or more determinations ina single sample, the use of two or more labels with differentelectrochemical properties in those circumstances permitting effectivedistinction between measurements relating to the respective species tobe determined (e.g. see Example 22). The compounds of the invention alsogive consistent electrochemical responses making them useful as internalcontrols in assays.

Example 20—Binding of Labels to Protein

Labels of the invention are attached to a peptide via a free amine of,for example, a lysine residue in the peptide. Attachment may beaccomplished conventional techniques including functionalisation of thelabelling compound to form an active NHS (N-hydroxysuccinimide) esterand reaction of the functionalised ester with the free amine group ofthe peptide.

Example 21—Binding of Labels to Particles

A biotin molecule is coupled to a label, for example a label as made inany of the above examples. The biotinylation can be carried out in anautomated oligonucleotide synthesiser or using standard laboratoryconditions by reaction of a ferrocenyl phosphoramidite label with NHSesters of biotin.

Paramagnetic streptavidin particles are washed ×3 (phosphate buffer) andmixed with biotinylated label, followed by incubation for 1 hour at roomtemperature with mixing. The particles are washed ×2 (phosphate buffer)and washed ×1 (PCR buffer). They are resuspended in final buffer (PCRbuffer). Following each wash step the supernatants are tested forelectrochemical signal, and if necessary washing is repeated until thesupernatants show no indication of free electrochemical label.

These particles are assayed at a range of concentrations to validatethat the observed electrochemical signal is attributable to the labelcoupled to the magnetic particles, using magnetic capture of theparticles and resuspension in a range of buffer volumes.

Example 22—Multiplex PCR Assay

The ferrocene compounds 2, 14, and 6 were converted in theircorresponding phosphoramidites using the procedures described herein.Two diferrocene labels were also converted to phosphoramidites, namely6-(bis-methylferrocenyl)amino)hexan-1-ol (‘di-1’) and6-(bis((1′-chloroferrocenyl) 1-methylferrocenyl)amino)hexan-1-ol(‘di-2’). Using standard solid phase coupling methodologies these fivephosphoramidites were then coupled at the 5′-end to five oligonucleotideprobes, each designed to detect a specific gene. The labels and probetargets were as follows:

Label Target Label Target 2 Internal control di-1 C. trachomatis gene 14S. aureus gene di-2 N. gonorrhoeae gene 6 T. vaginalis gene

PCR was performed on various samples using primers designed for the fivetarget genes listed in this table. The separate amplifications were themcombined to give a 5-plex mixture, and the five labelled probes werethen added to this mixture, together with T7 exonuclease. This mixturewas incubated and detection was performed essentially as set out inPearce et al. using screen-printed electrodes.

FIG. 1 shows six superimposed voltammograms: three performed on samplescontaining the various target genes (positive control), and threeperformed on blank samples (negative control):

The three negative control samples show no visible peaks between −0.5and +0.7 volts. In contrast, the three positive control samples eachshow five separate peaks. From left to right, these peaks correspond tolabels 2, 14, di-1, di-2, and 6. Thus the monoferrocene labels of theinvention are useful as labels in nucleic acid hybridisation assays,including multiplex assays, and they can be used also in combinationwith diferrocene labels.

Example 23—Reproducibility Experiment

3-(nonamethylferrocenylmethoxy)propan-1-ol (example compound 2) wasconjugated to an oligonucleotide using standard conditions. Theresulting probe was utilised at concentration of 5 μM in 52 separatePCRs amplifying 1000 copies of DNA. Electrochemical detection of theprobe yielded the current data in FIG. 2.

This data shows that the compounds of the invention give consistent,reproducible electrochemical signals. This makes the compounds of theinvention particularly useful in assays, for example as internalcontrols.

It will be understood that the invention is described above by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

The invention claimed is:
 1. A compound of formula IA′

wherein: each X substituent is independently selected from the groupconsisting of halo, vinyl, alkyl, cycloalkyl, SiR₃, SnR₃, PR₂, P(O)R₂,SR, S(O)R, SO₂R, aryl, heteroaryl, CHO, CO₂R, CN and CF₃; each R isindependently selected from the group consisting of alkyl, aryl,cycloalkyl, and heteroaryl; A is CH₂, B is O and c is 2; a is 0, 1, 2, 3or 4 and b is 0; and vinyl, alkyl, cycloalkyl, aryl and heteroaryl mayoptionally be substituted with 1, 2 or 3 substituents independentlyselected from the group consisting of unsubstituted OH, CN, fluorine,chlorine, bromine, and iodine, wherein F comprises a group selected fromthe group consisting of succinimidyl ester groups, phosphoramiditegroups, maleimide groups, biotin groups, and azide groups; and whereinsaid compound has an electrochemical potential of at least 450 mV. 2.The compound of claim 1 selected from the group consisting of:2-cyanoethyl-(3-(ferrocenylmethoxy)propan-1-ol)di-iso-propylphosphoramidite;2-cyanoethyl-(3-((1′-chloro)-ferrocenylmethoxy)propan-1-ol)di-iso-propylphosphoramidite;2-cyanoethyl-(3-((2-tert-butylthio)-ferrocenylmethoxy)propan-1-ol)di-iso-propylphosphoramidite;2-cyanoethyl-(3-((2-tert-butylsulfinyl)-ferrocenylmethoxy)propan-1-ol)di-iso-propylphosphoramidite;2-cyanoethyl-(3-((2-tert-butylsulfonyl)-ferrocenylmethoxy)propan-1-ol)di-iso-propylphosphoramidite;2-cyanoethyl-(3-((2-di-tert-butylphospinyl)-ferrocenylmethoxy)propan-1-ol)di-iso-propylphosphoramidite;2-cyanoethyl-(3-(2-tributylstannyl-ferrocenylmethoxy)propan-1-ol)di-iso-propylphosphoramidite;2-cyanoethyl-(3-(2-trimethylsilyl-ferrocenylmethoxy)propan-1-ol)di-iso-propylphosphoramidite;2-cyanoethyl-(3-(2-tributylsilyl-ferrocenylmethoxy)propan-1-ol)di-iso-propylphosphoramidite;2-cyanoethyl-(3-(2-trimethylstannyl-ferrocenylmethoxy)propan-1-ol)di-iso-propylphosphoramidite;2-cyanoethyl-(3-(2-vinyl-ferrocenylmethoxy)propan-1-ol)di-iso-propylphosphoramidite;2-cyanoethyl-(3-(2-iodo-ferrocenylmethoxy)propan-1-ol)di-iso-propylphosphoramidite;2-cyanoethyl-(2-(3-ferrocenylpropoxy)ethanol)di-iso-propylphosphoramidite;2-cyanoethyl-(2-(3-(2-tert-butylthio)-ferrocenylpropoxy)ethanol)di-iso-propylphosphoramidite;2-cyanoethyl-(2-(3-(2-tert-butylsulfinyl)-ferrocenylpropoxy)ethanol)di-iso-propylphosphoramidite;and2-cyanoethyl-(2-(3-(2-tert-butylsulfonyl)-ferrocenylpropoxy)ethanol)di-iso-propylphosphoramidite.3. The compound of claim 1 as an internal control in an electrochemicalassay.
 4. A method for the manufacture of the compound of claim 1, themethod comprising: reacting a compound of formula IA with afunctionalising compound to obtain a compound of formula IA′ of claim 1,wherein the compound of formula IA is of the following formula:

and X, a, b, c, A and B are as defined in claim
 1. 5. The compound ofclaim 1, according to formula IIA:

wherein: each X substituent is independently selected from halo, vinyl,alkyl, cycloalkyl, SiR₃, SnR₃, PR₂, P(O)R₂, SR, S(O)R, SO₂R, aryl,heteroaryl, CHO, CO₂R, CN and CF₃; A is CH₂, B is O and c is 2; a is 0,1, 2, 3 or 4; b is 0; and vinyl, alkyl, cycloalkyl, aryl and heteroarylmay optionally be substituted with 1, 2 or 3 substituents independentlyselected from unsubstituted OH, CN, fluorine, chlorine, bromine andiodine.